Detecting esophageal disorders

ABSTRACT

Provided herein is technology for esophageal disorder screening and particularly, but not exclusively, to methods, compositions, and related uses for detecting the presence of esophageal disorders (e.g., Barrett&#39;s esophagus, Barrett&#39;s esophageal dysplasia, etc.). In addition, the technology provides methods, compositions and related uses for distinguishing between Barrett&#39;s esophagus and Barrett&#39;s esophageal dysplasia, and between Barrett&#39;s esophageal low-grade dysplasia, Barrett&#39;s esophageal high-grade dysplasia, and esophageal adenocarcinoma within samples obtained through endoscopic brushing or nonendoscopic whole esophageal brushing or swabbing using a tethered device (e.g. such as a capsule sponge, balloon, or other device).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/550,703, filed Aug. 11, 2017, which is a 371 National Entryof International Patent Application No. PCT/US2016/023782, filed Mar.23, 2016, which claims priority to U.S. Provisional Patent ApplicationNo. 62/139,243, filed Mar. 27, 2015, each of which is incorporated byreference in its entirety.

FIELD OF INVENTION

Provided herein is technology for esophageal disorder screening andparticularly, but not exclusively, to methods, compositions, and relateduses for detecting the presence of esophageal disorders (e.g., Barrett'sesophagus, Barrett's esophageal dysplasia, etc.). In addition, thetechnology provides methods, compositions and related uses fordistinguishing between Barrett's esophagus and Barrett's esophagealdysplasia, and between Barrett's esophageal low-grade dysplasia,Barrett's esophageal high-grade dysplasia, and esophageal adenocarcinomawithin samples obtained through endoscopic brushing or nonendoscopicwhole esophageal brushing or swabbing using a tethered device (e.g. suchas a capsule sponge, balloon, or other device).

BACKGROUND

In Barrett's esophagus, healthy esophageal epithelium is replaced withmetaplastic columnar cells—the result, it is believed, of damage fromprolonged exposure of the esophagus to the refluxate of gastroesophagealreflux disease (GERD). The inherent risk of progression from Barrettesophagus to adenocarcinoma of the esophagus has been established.Histologically, this progression involves clear sequential stages frommetaplasia alone to low grade dysplasia, then to high grade dysplasia,and finally to adenocarcinoma.

The diagnosis of Barrett's esophagus without dysplasia does not lead tospecific therapy. However, when dysplasia is present, there is soundevidence that endoscopic ablation treatment can prevent subsequenttransformation to cancer. Such dysplasia is often endoscopicallyindistinguishable from Barrett's without dysplasia, and periodic randombiopsies with histological assessment is the current approach tosurveillance in patients with proven Barrett's.

Little evidence supports the assumption that antisecretory agents orantireflux surgery prevents the occurrence of adenocarcinoma or leads toregression of Barrett esophagus (see, e.g., Haag S, et al., GastrointestEndosc. August 1999; 50(2):229-40).

In the early to mid-1980s, histamine 2 (H2)-receptor antagonists werethe most commonly prescribed agents for treatment of GERD. However, anumber of studies were conducted with either cimetidine or ranitidine,and none documented regression of Barrett esophagus.

In the late 1980s, proton pump inhibitors (PPIs) were introduced andproved to be much more efficacious at reducing gastric acid secretion.Even so, the supposition that better acid suppression could induceBarrett's esophagus regression was met with optimism, and studies onthis to date have been inconclusive. Only 2 of 7 investigatorsdemonstrated some regression. Most were unable to detect any regression,despite documentation of complete normalization of esophageal pH by pHtesting.

Currently, the indications for medical therapy in Barrettesophagus—control of symptoms and healing of esophageal mucosa—are thesame as those for GERD.

Barrett's Esophagus is a precursor lesion for most esophagealadenocarcinomas which is a malignancy with rapidly rising incidence andpersistently poor outcomes. As above, early detection of esophagealadenocarcinoma has been shown to be associated with earlier stage andincreased survival. And, detection of dysplasia with subsequentendoscopic ablation can prevent esophageal adenocarcinoma.

Improved methods for detecting Barrett's esophagus and related disorders(e.g., Barrett's esophageal dysplasia) are clearly needed.

SUMMARY

Methylated DNA has been studied as a potential class of biomarkers inthe tissues of most tumor types. In many instances, DNAmethyltransferases add a methyl group to DNA atcytosine-phosphate-guanine (CpG) island sites as an epigenetic controlof gene expression. In a biologically attractive mechanism, acquiredmethylation events in promoter regions of tumor suppressor genes arethought to silence expression, thus contributing to oncogenesis. DNAmethylation may be a more chemically and biologically stable diagnostictool than RNA or protein expression (Laird (2010) Nat Rev Genet 11:191-203). Furthermore, in other cancers like sporadic colon cancer,methylation markers offer excellent specificity and are more broadlyinformative and sensitive than are individual DNA mutations (Zou et al(2007) Cancer Epidemiol Biomarkers Prev 16: 2686-96).

Analysis of CpG islands has yielded important findings when applied toanimal models and human cell lines. For example, Zhang and colleaguesfound that amplicons from different parts of the same CpG island mayhave different levels of methylation (Zhang et al. (2009) PLoS Genet 5:e1000438). Further, methylation levels were distributed bi-modallybetween highly methylated and unmethylated sequences, further supportingthe binary switch-like pattern of DNA methyltransferase activity (Zhanget al. (2009) PLoS Genet 5: e1000438). Analysis of murine tissues invivo and cell lines in vitro demonstrated that only about 0.3% of highCpG density promoters (HCP, defined as having >7% CpG sequence within a300 base pair region) were methylated, whereas areas of low CpG density(LCP, defined as having <5% CpG sequence within a 300 base pair region)tended to be frequently methylated in a dynamic tissue-specific pattern(Meissner et al. (2008) Nature 454: 766-70). HCPs include promoters forubiquitous housekeeping genes and highly regulated developmental genes.Among the HCP sites methylated at >50% were several established markerssuch as Wnt 2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature454: 766-70).

Accordingly, provided herein is technology for esophageal disorderscreening (e.g., surveilling) and particularly, but not exclusively, tomethods, compositions, and related uses for detecting the presence ofesophageal disorders (e.g., Barrett's esophagus, Barrett's esophagealdysplasia, etc.). In addition, the technology provides methods,compositions and related uses for distinguishing between Barrett'sesophagus and Barrett's esophageal dysplasia, and between Barrett'sesophageal low-grade dysplasia, Barrett's esophageal high-gradedysplasia, and esophageal adenocarcinoma within samples obtained throughendoscopic brushing or nonendoscopic whole esophageal brushing orswabbing using a tethered device (e.g. such as a capsule sponge,balloon, or other device).

Indeed, experiments conducted during the course of developing thistechnology compared the methylation state of DNA markers from esophagealtissue of subjects having Barrett's esophagus to the methylation stateof the same DNA markers from control subjects (e.g., normal tissue forthe respective tissue type), and to the methylation state of the sameDNA markers from subjects having Barrett's esophagus dysplasia (see,Examples 1 and 5).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Tables 1, 7 and/or 8) capable ofclassifying Barrett's esophagus (BE) versus control (e.g., normal tissuefor the respective tissue type) within esophageal tissue (see, Examples1, 2 and 5).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Tables 2, 3, 5, and/or 6)capable of classifying BE versus Barrett's esophagus related dyspasia(BED) within esophageal tissue (see, Examples 1, 3 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of predictingBarrett's esophagus related low-grade dyspasia (BE-LGD), Barrett'sesophagus related dyspasia high-grade dysplasia (BE-HGD), and esophagealadenocarcinoma (EAC) within samples obtained through whole esophagealswabbing or brushing (see, Examples 1 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of classifyingBE versus BED within samples obtained through whole esophageal swabbingor brushing (see, Examples 1 and 4).

As described herein, the technology provides a number of methylated DNAmarkers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, 10, 15, 25,50, 100, 150, 180, 190, 194 markers) with high discrimination foresophageal disorders (e.g., BE, BED, BE-LGD, BE-HGD, EAC). Experimentsapplied a selection filter to candidate markers to identify markers thatprovide a high signal to noise ratio and a low background level toprovide high specificity, e.g., when assaying media (e.g., esophagealtissue) for purposes of screening or diagnosis (e.g., cancer screeningor diagnosis).

In some embodiments, the technology is related to assessing the presenceof and methylation state of one or more of the markers identified hereinin a biological sample. These markers comprise one or moredifferentially methylated regions (DMR) as discussed herein, e.g., asprovided in Tables 1, 2, 3, 5, 6, 7 and 8. Methylation state is assessedin embodiments of the technology. As such, the technology providedherein is not restricted in the method by which a gene's methylationstate is measured. For example, in some embodiments the methylationstate is measured by a genome scanning method. For example, one methodinvolves restriction landmark genomic scanning (Kawai et al. (1994) Mol.Cell. Biol. 14: 7421-7427) and another example involvesmethylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997)Cancer Res. 57: 594-599). In some embodiments, changes in methylationpatterns at specific CpG sites are monitored by digestion of genomic DNAwith methylation-sensitive restriction enzymes followed by Southernanalysis of the regions of interest (digestion-Southern method). In someembodiments, analyzing changes in methylation patterns involves aPCR-based process that involves digestion of genomic DNA withmethylation-sensitive restriction enzymes prior to PCR amplification(Singer-Sam et al. (1990) Nucl. Acids Res. 18: 687). In addition, othertechniques have been reported that utilize bisulfite treatment of DNA asa starting point for methylation analysis. These includemethylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad.Sci. USA 93: 9821-9826) and restriction enzyme digestion of PCR productsamplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl.Acids Res. 24: 5058-5059; and Xiong and Laird (1997) Nucl. Acids Res.25: 2532-2534). PCR techniques have been developed for detection of genemutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and quantification of allelic-specific expression (Szabo andMann (1995) Genes Dev. 9: 3097-3108; and Singer-Sam et al. (1992) PCRMethods Appl. 1: 160-163). Such techniques use internal primers, whichanneal to a PCR-generated template and terminate immediately 5′ of thesingle nucleotide to be assayed. Methods using a “quantitative Ms-SNuPEassay” as described in U.S. Pat. No. 7,037,650 are used in someembodiments.

Upon evaluating a methylation state, the methylation state is oftenexpressed as the fraction or percentage of individual strands of DNAthat is methylated at a particular site (e.g., at a single nucleotide,at a particular region or locus, at a longer sequence of interest, e.g.,up to a ˜100-bp, 200-bp, 500-bp, 1000-bp subsequence of a DNA or longer)relative to the total population of DNA in the sample comprising thatparticular site. Traditionally, the amount of the unmethylated nucleicacid is determined by PCR using calibrators. Then, a known amount of DNAis bisulfite treated and the resulting methylation-specific sequence isdetermined using either a real-time PCR or other exponentialamplification, e.g., a QuARTS assay (e.g., as provided by U.S. Pat. No.8,361,720; and U.S. Pat. Appl. Pub. Nos. 2012/0122088 and 2012/0122106).

For example, in some embodiments methods comprise generating a standardcurve for the unmethylated target by using external standards. Thestandard curve is constructed from at least two points and relates thereal-time Ct value for unmethylated DNA to known quantitative standards.Then, a second standard curve for the methylated target is constructedfrom at least two points and external standards. This second standardcurve relates the Ct for methylated DNA to known quantitative standards.Next, the test sample Ct values are determined for the methylated andunmethylated populations and the genomic equivalents of DNA arecalculated from the standard curves produced by the first two steps. Thepercentage of methylation at the site of interest is calculated from theamount of methylated DNAs relative to the total amount of DNAs in thepopulation, e.g., (number of methylated DNAs)/(the number of methylatedDNAs+number of unmethylated DNAs)×100.

Also provided herein are compositions and kits for practicing themethods. For example, in some embodiments, reagents (e.g., primers,probes) specific for one or more markers are provided alone or in sets(e.g., sets of primers pairs for amplifying a plurality of markers).Additional reagents for conducting a detection assay may also beprovided (e.g., enzymes, buffers, positive and negative controls forconducting QuARTS, PCR, sequencing, bisulfite, or other assays). In someembodiments, the kits containing one or more reagent necessary,sufficient, or useful for conducting a method are provided. Alsoprovided are reactions mixtures containing the reagents. Furtherprovided are master mix reagent sets containing a plurality of reagentsthat may be added to each other and/or to a test sample to complete areaction mixture.

In some embodiments, the technology described herein is associated witha programmable machine designed to perform a sequence of arithmetic orlogical operations as provided by the methods described herein. Forexample, some embodiments of the technology are associated with (e.g.,implemented in) computer software and/or computer hardware. In oneaspect, the technology relates to a computer comprising a form ofmemory, an element for performing arithmetic and logical operations, anda processing element (e.g., a microprocessor) for executing a series ofinstructions (e.g., a method as provided herein) to read, manipulate,and store data. In some embodiments, a microprocessor is part of asystem for determining a methylation state (e.g., of one or more DMR,e.g., DMR 1-78 as provided in Table 1; DMR 3, 5, 30, 33, 43, 58, 77 and79-128 as provided in Table 2; DMR 77, 27, 193, 90, 92, 101 and 129-134as provided in Table 3; DMR 77, 90 and 135 as provided in Table 5; DMR136-187 as provided in Table 6; DMR 21 and 188-192 as provided in Table7; DMR 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136,141, 142, 144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175,178, 179, 181, 185, 187, and 193-229 as provided in Table 8); comparingmethylation states (e.g., of one or more DMR, e.g., DMR 1-78 as providedin Table 1; DMR 3, 5, 30, 33, 43, 58, 77 and 79-128 as provided in Table2; DMR 77, 27, 193, 90, 92, 101 and 129-134 as provided in Table 3; DMR77, 90 and 135 as provided in Table 5; DMR 136-187 as provided in Table6; DMR 21 and 188-192 as provided in Table 7; DMR 2-4, 6, 7, 14, 30, 77,80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149,152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and193-229 as provided in Table 8); generating standard curves; determininga Ct value; calculating a fraction, frequency, or percentage ofmethylation (e.g., of one or more DMR, e.g., DMR 1-78 as provided inTable 1; DMR 3, 5, 30, 33, 43, 58, 77 and 79-128 as provided in Table 2;DMR 77, 27, 193, 90, 92, 101 and 129-134 as provided in Table 3; DMR 77,90 and 135 as provided in Table 5; DMR 136-187 as provided in Table 6;DMR 21 and 188-192 as provided in Table 7; DMR 2-4, 6, 7, 14, 30, 77,80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144, 146, 148-149,152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181, 185, 187, and193-229 as provided in Table 8); identifying a CpG island; determining aspecificity and/or sensitivity of an assay or marker; calculating an ROCcurve and an associated AUC; sequence analysis; all as described hereinor is known in the art.

In some embodiments, a microprocessor or computer uses methylation statedata in an algorithm to predict a site of a cancer.

In some embodiments, a software or hardware component receives theresults of multiple assays and determines a single value result toreport to a user that indicates a cancer risk based on the results ofthe multiple assays (e.g., determining the methylation state of multipleDMR, e.g., as provided in Tables 1, 2, 3, 5, 6, 7, 8). Relatedembodiments calculate a risk factor based on a mathematical combination(e.g., a weighted combination, a linear combination) of the results frommultiple assays, e.g., determining the methylation states of multiplemarkers (such as multiple DMR, e.g., as provided in Tables 1, 2, 3, 5,6, 7, 8). In some embodiments, the methylation state of a DMR defines adimension and may have values in a multidimensional space and thecoordinate defined by the methylation states of multiple DMR is aresult, e.g., to report to a user, e.g., related to an esophagealdisorder risk (e.g., risk of BE, BED, BE-LGD, BE-HGD, EAC).

Some embodiments comprise a storage medium and memory components. Memorycomponents (e.g., volatile and/or nonvolatile memory) find use instoring instructions (e.g., an embodiment of a process as providedherein) and/or data (e.g., a work piece such as methylationmeasurements, sequences, and statistical descriptions associatedtherewith). Some embodiments relate to systems also comprising one ormore of a CPU, a graphics card, and a user interface (e.g., comprisingan output device such as display and an input device such as akeyboard).

Programmable machines associated with the technology compriseconventional extant technologies and technologies in development or yetto be developed (e.g., a quantum computer, a chemical computer, a DNAcomputer, an optical computer, a spintronics based computer, etc.).

In some embodiments, the technology comprises a wired (e.g., metalliccable, fiber optic) or wireless transmission medium for transmittingdata. For example, some embodiments relate to data transmission over anetwork (e.g., a local area network (LAN), a wide area network (WAN), anad-hoc network, the internet, etc.). In some embodiments, programmablemachines are present on such a network as peers and in some embodimentsthe programmable machines have a client/server relationship.

In some embodiments, data are stored on a computer-readable storagemedium such as a hard disk, flash memory, optical media, a floppy disk,etc.

In some embodiments, the technology provided herein is associated with aplurality of programmable devices that operate in concert to perform amethod as described herein. For example, in some embodiments, aplurality of computers (e.g., connected by a network) may work inparallel to collect and process data, e.g., in an implementation ofcluster computing or grid computing or some other distributed computerarchitecture that relies on complete computers (with onboard CPUs,storage, power supplies, network interfaces, etc.) connected to anetwork (private, public, or the internet) by a conventional networkinterface, such as Ethernet, fiber optic, or by a wireless networktechnology.

For example, some embodiments provide a computer that includes acomputer-readable medium. The embodiment includes a random access memory(RAM) coupled to a processor. The processor executes computer-executableprogram instructions stored in memory. Such processors may include amicroprocessor, an ASIC, a state machine, or other processor, and can beany of a number of computer processors, such as processors from IntelCorporation of Santa Clara, Calif. and Motorola Corporation ofSchaumburg, Ill. Such processors include, or may be in communicationwith, media, for example computer-readable media, which storesinstructions that, when executed by the processor, cause the processorto perform the steps described herein.

Embodiments of computer-readable media include, but are not limited to,an electronic, optical, magnetic, or other storage or transmissiondevice capable of providing a processor with computer-readableinstructions. Other examples of suitable media include, but are notlimited to, a floppy disk, CD-ROM, DVD, magnetic disk, memory chip, ROM,RAM, an ASIC, a configured processor, all optical media, all magnetictape or other magnetic media, or any other medium from which a computerprocessor can read instructions. Also, various other forms ofcomputer-readable media may transmit or carry instructions to acomputer, including a router, private or public network, or othertransmission device or channel, both wired and wireless. Theinstructions may comprise code from any suitable computer-programminglanguage, including, for example, C, C++, C #, Visual Basic, Java,Python, Perl, and JavaScript.

Computers are connected in some embodiments to a network. Computers mayalso include a number of external or internal devices such as a mouse, aCD-ROM, DVD, a keyboard, a display, or other input or output devices.Examples of computers are personal computers, digital assistants,personal digital assistants, cellular phones, mobile phones, smartphones, pagers, digital tablets, laptop computers, internet appliances,and other processor-based devices. In general, the computers related toaspects of the technology provided herein may be any type ofprocessor-based platform that operates on any operating system, such asMicrosoft Windows, Linux, UNIX, Mac OS X, etc., capable of supportingone or more programs comprising the technology provided herein. Someembodiments comprise a personal computer executing other applicationprograms (e.g., applications). The applications can be contained inmemory and can include, for example, a word processing application, aspreadsheet application, an email application, an instant messengerapplication, a presentation application, an Internet browserapplication, a calendar/organizer application, and any other applicationcapable of being executed by a client device.

All such components, computers, and systems described herein asassociated with the technology may be logical or virtual.

Accordingly, provided herein is technology related to a method ofscreening for BE in a sample obtained from a subject, the methodcomprising assaying a methylation state of a marker in a sample obtainedfrom a subject; and identifying the subject as having BE when themethylation state of the marker is different than a methylation state ofthe marker assayed in a subject that does not have BE, wherein themarker comprises one or more bases in a differentially methylated region(DMR) selected from a group consisting of DMR 1-78 as provided in Table1 and/or DMR 21 and 188-193 as provided in Table 7 and/or; DMR 2-4, 6,7, 14, 30, 77, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142, 144,146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179, 181,185, 187, and 193-229 as provided in Table 8.

Provided herein is technology related to a method of distinguishingbetween BE and BED in a sample obtained from a subject, the methodcomprising assaying a methylation state of a marker in a sample obtainedfrom a subject; and identifying the subject as having BE when themethylation state of the marker is similar to the methylation state ofthe marker assayed in a subject that has BE or identifying the subjectas having BED when the methylation state of the marker is similar to themethylation state of the marker assayed in a subject that has BED,wherein the marker comprises one or more bases in a differentiallymethylated region (DMR) selected from a group consisting of DMR 3, 5,30, 33, 43, 77 and 79-128 as provided in Table 2, DMR 77, 27, 193, 90,92, 101, 129-134 as provided in Table 3, DMR 77, 90 and 135 as providedin Table 5, and/or DMR 136-187 as provided in Table 6.

Provided herein is technology related to a method of distinguishingbetween BE-LGD, BE-HGD, and BE-EAC in a sample obtained from a subject,the method comprising assaying a methylation state of a marker in asample obtained from a subject; and identifying the subject as havingBE-LGD when the methylation state of the marker is similar to themethylation state of the marker assayed in a subject that has BE-LGD,identifying the subject as having BE-HGD when the methylation state ofthe marker is similar to the methylation state of the marker assayed ina subject that has BE-HGD, or identifying the subject as having EAC whenthe methylation state of the marker is similar to the methylation stateof the marker assayed in a subject that has EAC, wherein the markercomprises one or more bases in a differentially methylated region (DMR)selected from a group consisting of DMR 77, 90 and 135 as provided inTable 5.

The technology is not limited in the methylation state assessed. In someembodiments assessing the methylation state of the marker in the samplecomprises determining the methylation state of one base. In someembodiments, assaying the methylation state of the marker in the samplecomprises determining the extent of methylation at a plurality of bases.Moreover, in some embodiments the methylation state of the markercomprises an increased methylation of the marker relative to a normalmethylation state of the marker. In some embodiments, the methylationstate of the marker comprises a decreased methylation of the markerrelative to a normal methylation state of the marker. In someembodiments the methylation state of the marker comprises a differentpattern of methylation of the marker relative to a normal methylationstate of the marker.

Furthermore, in some embodiments the marker is a region of 100 or fewerbases, the marker is a region of 500 or fewer bases, the marker is aregion of 1000 or fewer bases, the marker is a region of 5000 or fewerbases, or, in some embodiments, the marker is one base. In someembodiments the marker is in a high CpG density promoter.

The technology is not limited by sample type. In some embodiments, thesample is esophageal tissue obtained through whole esophageal swabbingor brushing (see, Example 1 and Table 5) (see, Example 5 and Table 8).In some embodiments, the sample is esophageal tissue obtained throughuse a sponge capsule device (see, Example 5 and Table 8). For example,in some embodiments the sample is a stool sample, a tissue sample (e.g.,esophageal tissue, stomach tissue, pancreatic tissue, bile duct/livertissue, and colorectal tissue), a blood sample (e.g., plasma, serum,whole blood), an excretion, or a urine sample.

Furthermore, the technology is not limited in the method used todetermine methylation state. In some embodiments the assaying comprisesusing methylation specific polymerase chain reaction, nucleic acidsequencing, mass spectrometry, methylation specific nuclease, mass-basedseparation, or target capture. In some embodiments, the assayingcomprises use of a methylation specific oligonucleotide. In someembodiments, the technology uses massively parallel sequencing (e.g.,next-generation sequencing) to determine methylation state, e.g.,sequencing-by-synthesis, real-time (e.g., single-molecule) sequencing,bead emulsion sequencing, nanopore sequencing, etc.

The technology provides reagents for detecting a DMR, e.g., in someembodiments are provided a set of oligonucleotides comprising thesequences provided by SEQ ID NO: 1-50. In some embodiments are providedan oligonucleotide comprising a sequence complementary to a chromosomalregion having a base in a DMR, e.g., an oligonucleotide sensitive tomethylation state of a DMR.

The technology provides various panels of markers, e.g., in someembodiments the marker comprises a chromosomal region having anannotation that is provided in Tables 1, 2, 3, 5, 6, 7 and/or 8, andthat comprises the marker (see, Tables 1, 2, 3, 5, 6, 7, 8). Inaddition, embodiments provide a method of analyzing a DMR from Tables 1,2, 3, 5, 6, 7 and/or 8 that one or more of DMR Nos. 1-229.

Kit embodiments are provided, e.g., a kit comprising a bisulfitereagent; and a control nucleic acid comprising a sequence from a DMRselected from a group consisting of DMR 1-194 (from Tables 1, 2, 3, 5,6, 7, and/or 8) and having a methylation state associated with a subjectwho does not have an esophageal disorder (e.g., a subject that does nothave BE, BED, BE-LGD, BE-HGD, and EAC). In some embodiments, kitscomprise a bisulfite reagent and an oligonucleotide as described herein.In some embodiments, kits comprise a bisulfite reagent; and a controlnucleic acid comprising a sequence from a DMR selected from a groupconsisting of DMR 1-194 (from Tables 1, 2, 3, 5, 6, 7, 8) and having amethylation state associated with a subject who has an esophagealdisorder (e.g., a subject who has BE) (e.g., a subject who has BED,BE-LGD, BE-HGD, EAC). Some kit embodiments comprise a sample collectorfor obtaining a sample from a subject (e.g., a stool sample); reagentsfor isolating a nucleic acid from the sample; a bisulfite reagent; andan oligonucleotide as described herein.

The technology is related to embodiments of compositions (e.g., reactionmixtures). In some embodiments are provided a composition comprising anucleic acid comprising a DMR and a bisulfite reagent. Some embodimentsprovide a composition comprising a nucleic acid comprising a DMR and anoligonucleotide as described herein. Some embodiments provide acomposition comprising a nucleic acid comprising a DMR and amethylation-sensitive restriction enzyme. Some embodiments provide acomposition comprising a nucleic acid comprising a DMR and a polymerase.

Additional related method embodiments are provided for screening for anesophageal disorder (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a sampleobtained from a subject, e.g., a method comprising determining amethylation state of a marker in the sample comprising a base in a DMRthat is one or more of DMR 1-229 (from Table 1, 2, 3, 5, 6, 7, 8);comparing the methylation state of the marker from the subject sample toa methylation state of the marker from a normal control sample from asubject who does not have an esophageal disorder; and determining aconfidence interval and/or a p value of the difference in themethylation state of the subject sample and the normal control sample.In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%,99%, 99.5%, 99.9% or 99.99% and the p value is 0.1, 0.05, 0.025, 0.02,0.01, 0.005, 0.001, or 0.0001. Some embodiments of methods provide stepsof reacting a nucleic acid comprising a DMR with a bisulfate reagent toproduce a bisulfate-reacted nucleic acid; sequencing thebisulfate-reacted nucleic acid to provide a nucleotide sequence of thebisulfate-reacted nucleic acid; comparing the nucleotide sequence of thebisulfate-reacted nucleic acid with a nucleotide sequence of a nucleicacid comprising the DMR from a subject who does not have a cancer toidentify differences in the two sequences; and identifying the subjectas having a neoplasm when a difference is present.

Systems for screening for an esophageal disorder (e.g., BE, BED, BE-LGD,BE-HGD, EAC) in a sample obtained from a subject are provided by thetechnology. Exemplary embodiments of systems include, e.g., a system forscreening for an esophageal disorder in a sample obtained from asubject, the system comprising an analysis component configured todetermine the methylation state of a sample, a software componentconfigured to compare the methylation state of the sample with a controlsample or a reference sample methylation state recorded in a database,and an alert component configured to alert a user of an esophagealdisorder-associated methylation state (e.g., a methylation state for noesophageal disorder; a methylation state for BE; a methylation state forBED; a methylation state for BE-LGD; a methylation state for BE-HGD; amethylation state for EAC). An alert is determined in some embodimentsby a software component that receives the results from multiple assays(e.g., determining the methylation states of multiple markers, e.g.,DMR, e.g., as provided in Table 1, 2, 3, 5, 6, 7, and/or 8) andcalculating a value or result to report based on the multiple results.Some embodiments provide a database of weighted parameters associatedwith each DMR provided herein for use in calculating a value or resultand/or an alert to report to a user (e.g., such as a physician, nurse,clinician, etc.). In some embodiments all results from multiple assaysare reported and in some embodiments one or more results are used toprovide a score, value, or result based on a composite of one or moreresults from multiple assays that is indicative of an esophagealdisorder risk in a subject (e.g., risk indicative for BE; riskindicative for BED; risk indicative for BE-LGD; risk indicative forBE-HGD; risk indicative for EAC).

In some embodiments of systems, a sample comprises a nucleic acidcomprising a DMR. In some embodiments the system further comprises acomponent for isolating a nucleic acid, a component for collecting asample such as a component for collecting a stool sample. In someembodiments, the system comprises nucleic acid sequences comprising aDMR. In some embodiments the database comprises nucleic acid sequencesfrom subjects who do not have an esophageal disorder. Also provided arenucleic acids, e.g., a set of nucleic acids, each nucleic acid having asequence comprising a DMR. In some embodiments the set of nucleic acidswherein each nucleic acid has a sequence from a subject who does nothave an esophageal disorder. Related system embodiments comprise a setof nucleic acids as described and a database of nucleic acid sequencesassociated with the set of nucleic acids. Some embodiments furthercomprise a bisulfite reagent. And, some embodiments further comprise anucleic acid sequencer.

In certain embodiments, methods for detecting Barrett's esophagus in asample obtained from a subject are provided, comprising a) obtaining asample comprising DNA from a subject; b) treating the obtained DNA witha reagent which selectively modifies unmethylated cytosine residues inthe obtained DNA to produce modified residues but which does not modifymethylated cytosine residues; c) determining the methylation level ofone or more DNA methylation markers in the DNA having undergone thetreating of step b), wherein one or more DNA methylation markerscomprises a base in a differentially methylated region (DMR) as providedby DMR Nos. 1-78, 80, 82-86, 88, 90-102, 108, 122, 135, 136, 141, 142,144, 146, 148-149, 152, 154, 156, 164, 166, 171, 173, 175, 178, 179,181, 185, and 187-229 d) comparing the determined methylation level ofthe one or more DNA methylation markers with methylation levelreferences for the one or more DNA methylation markers for subjects: i)who do not have Barrett's esophagus to identify differences in the twosequences, and ii) who do not have Barrett's esophageal dysplasia toidentify differences in the two sequences; and e) identifying thesubject as having Barrett's esophagus when differences in i) and ii) arepresent.

In certain embodiments, methods for detecting Barrett's esophagealdysplasia in a sample obtained from a subject are provided, comprisinga) obtaining a sample comprising DNA from a subject; b) treating theobtained DNA with a reagent which selectively modifies unmethylatedcytosine residues in the obtained DNA to produce modified residues butwhich does not modify methylated cytosine residues; c) determining themethylation level of one or more DNA methylation markers in the DNAhaving undergone the treating of step b), wherein one or more DNAmethylation markers comprises a base in a differentially methylatedregion (DMR) as provided by DMR Nos. 3, 5, 30, 33, 43, 58, 77, 79-187,d) comparing the determined methylation level of the one or more DNAmethylation markers with methylation level references for the one ormore DNA methylation markers for subjects: i) who have Barrett'sesophagus to identify differences in the two sequences, and ii) who donot have Barrett's esophageal dysplasia to identify differences in thetwo sequences; and e) identifying the subject as having Barrett'sesophageal dysplasia when differences in i) and ii) are present.

In certain embodiments, methods for detecting Barrett's esophageallow-grade dysplasia in a sample obtained from a subject are provided,comprising a) obtaining a sample comprising DNA from a subject; b)treating the obtained DNA with a reagent which selectively modifiesunmethylated cytosine residues in the obtained DNA to produce modifiedresidues but which does not modify methylated cytosine residues; c)determining the methylation level of one or more DNA methylation markersin the DNA having undergone the treating of step b), wherein one or moreDNA methylation markers comprises a base in a differentially methylatedregion (DMR) as provided by DMR Nos. 77, 90 and 135, d) comparing thedetermined methylation level of the one or more DNA methylation markerswith methylation level references for the one or more DNA methylationmarkers for subjects: i) who do not have Barrett's esophageal low-gradedysplasia to identify differences in the two sequences, ii) who do nothave Barrett's esophageal dysplasia to identify differences in the twosequences, iii) who have Barrett's esophageal high-grade dysplasia toidentify differences in the two sequences, and iv) who have esophagealadenocarcinoma to identify differences in the two sequences; and e)identifying the subject as having Barrett's esophageal low-gradedysplasia when differences in i), ii), iii, and iv) are present.

In certain embodiments, methods for detecting Barrett's esophagealhigh-grade dysplasia in a sample obtained from a subject are provided,comprising a) obtaining a sample comprising DNA from a subject; b)treating the obtained DNA with a reagent which selectively modifiesunmethylated cytosine residues in the obtained DNA to produce modifiedresidues but which does not modify methylated cytosine residues; c)determining the methylation level of one or more DNA methylation markersin the DNA having undergone the treating of step b), wherein one or moreDNA methylation markers comprises a base in a differentially methylatedregion (DMR) as provided by DMR Nos. 77, 90 and 135, d) comparing thedetermined methylation level of the one or more DNA methylation markerswith methylation level references for the one or more DNA methylationmarkers for subjects: i) who do not have Barrett's esophageal high-gradedysplasia to identify differences in the two sequences, ii) who do nothave Barrett's esophageal dysplasia to identify differences in the twosequences, iii) who have Barrett's esophageal low-grade dysplasia toidentify differences in the two sequences, and iv) who have esophagealadenocarcinoma to identify differences in the two sequences; and e)identifying the subject as having Barrett's esophageal high-gradedysplasia when differences in i), ii), iii, and iv) are present.

In certain embodiments, methods for detecting esophageal adenocarcinomain a sample obtained from a subject are provided, comprising a)obtaining a sample comprising DNA from a subject; b) treating theobtained DNA with a reagent which selectively modifies unmethylatedcytosine residues in the obtained DNA to produce modified residues butwhich does not modify methylated cytosine residues c) determining themethylation level of one or more DNA methylation markers in the DNAhaving undergone the treating of step b), wherein one or more DNAmethylation markers comprises a base in a differentially methylatedregion (DMR) as provided by DMR Nos. 77, 90 and 135, d) comparing thedetermined methylation level of the one or more DNA methylation markerswith methylation level references for the one or more DNA methylationmarkers for subjects: i) who do not have esophageal adenocarcinoma toidentify differences in the two sequences, ii) who do not have Barrett'sesophageal dysplasia to identify differences in the two sequences, iii)who have Barrett's esophageal low-grade dysplasia to identifydifferences in the two sequences, and iv) who have Barrett's esophagealhigh-grade dysplasia to identify differences in the two sequences; ande) identifying the subject as having esophageal adenocarcinoma whendifferences in i), ii), iii, and iv) are present.

In some embodiments, a determination of elevated methylation in one ormore of the DNA methylation markers comprises a determination of alteredmethylation within a region selected from the group consisting of a CpGisland and a CpG island shore.

In some embodiments, a determination of elevated methylation within theCpG island or CpG shore comprises elevated methylation within a codingregion or a regulatory region of the DNA methylation marker.

In some embodiments, the determining the methylation level of one ormore DNA methylation markers in the DNA having undergone the treating ofstep b) comprises determining the methylation score and/or themethylation frequency of the one or more DNA methylation markers. Insome embodiments, the treating of step b) is accomplished throughbisulfite modification of the obtained DNA.

In some embodiments, the determining the methylation level of one ormore DNA methylation markers in the DNA having undergone the treating ofstep b) is achieved by a technique selected from the group consisting ofmethylation-specific PCR, quantitative methylation-specific PCR,methylation-sensitive DNA restriction enzyme analysis, quantitativebisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

In some embodiments, the sample comprises esophageal tissue. In someembodiments, the esophageal tissue is obtained through endoscopicbrushing or nonendoscopic whole esophageal brushing or swabbing using atethered device (e.g. such as a capsule sponge, balloon, or otherdevice).

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Positivity rates of a 3-marker panel (DIO3, MAX20.218, NDRG4)(Table 5) in tissue DNA from BE subgroups without dysplasia and withdifferent seventies of dysplasia (Examples I, II, III and IV).

FIG. 2: Methylated DNA marker levels (PCR copies/20 ng DNA) in BE casesand normal (Nl) controls from Phase 2 (endoscopic brush study) (ExampleV).

FIG. 3: Hit Matrix of Top Methylated DNA markers from Phase 2highlighting complementarity (endoscopic brush study) (Example V).

FIG. 4: Methylated DNA marker levels (PCR copies/30 ng DNA) in BE casesand normal (Nl) controls from Phase 3 (capsule sponge study) (ExampleV).

DETAILED DESCRIPTION

Barrett's esophagus (BE) is the strongest risk factor for and only knownprecursor for esophageal adenocarcinoma (EAC), a lethal malignancy withpoor survival (<20% at 5 years) when detected after the onset ofsymptoms (see, Nelsen E M, et al., The Surgical clinics of North America2012; 92:1135-54). The incidence of esophageal adenocarcinoma hasincreased by almost 600% in the last three decades in the population(see, Hur C, et al., Cancer 2013; 119:1149-58). BE progresses to EACthrough a step-wise pathway from no dysplasia, to low grade dysplasia(LGD) to high grade dysplasia (HGD) to carcinoma. This metaplasia todysplasia to carcinoma sequence has prompted several nationalgastroenterology societies to recommend screening for BE in high risksubjects with multiple risk factors followed by endoscopic surveillance(depending on the grade of dysplasia) to detect the development ofdysplasia or carcinoma at an early stage (see, Spechler S J, et al.,Gastroenterology 2011; 140:e18-52; Wang K K, et al., Am J Gastroenterol2008; 103:788-97; Fitzgerald R C, et al., Gut 2014; 63:7-42). Endoscopictreatments of LGD, HGD and early carcinoma have been developed and shownto be effective in reducing the incidence of carcinoma and improvingsurvival in BE subjects (see, e.g., Prasad G A, et al., Gastroenterology2007; 132:1226-33; Prasad G A, et al., Gastroenterology 2009; Shaheen NJ, et al., N Engl J Med 2009; 360:2277-88; Phoa K N, et al., JAMA 2014;311:1209-17).

Screening for BE is currently performed using conventional sedatedendoscopy (sEGD) which reveals the replacement of the normal squamouslining of the esophagus by metaplastic columnar epithelium in subjectswith BE. However sedated endoscopy is expensive with both direct andindirect costs and not suitable for widespread application. It is alsoassociated with potential complications (see, Sami S S, et al., Clinicalgastroenterology and hepatology: the official clinical practice journalof the American Gastroenterological Association 2015; 13:623-34). Othertechniques such as unsedated transnasal endoscopy (uTNE) have comparableaccuracy to sEGD with lower cost, but continue to be poorly regarded asa widely applicable tool by providers (see, Sami S S, et al., TheAmerican journal of gastroenterology 2015; 110:148-58; Peery A F, etal., Gastrointestinal endoscopy 2012; 75:945-953 e2; Atkinson M, et al.,Gastroenterology & hepatology 2007; 4:426-7). Despite adequate access tothe uTNE device the utilization of uTNE by referring physicians remainslimited (see, Atkinson M, et al., The American journal ofgastroenterology 2008; 103:92-7). The absence of accurate riskstratification tools to determine BE risk and target screening effortsare additional limitations to a widely applicable BE screening (see,Sami S S, et al., Clinical gastroenterology and hepatology: the officialclinical practice journal of the American GastroenterologicalAssociation 2015; 13:623-34).

Endoscopic detection of dysplasia is currently performed using fourquadrant random biopsies every 1-2 cm of the BE segment in addition tocareful inspection of the BE segment with high resolution white lightimaging and advanced imaging techniques. While this has been recommendedby GI societies (see, e.g., Spechler S J, et al., Gastroenterology 2011;140:e18-52; Wang K K, et al., Am J Gastroenterol 2008; 103:788-97;Fitzgerald R C, et al., Gut 2014; 63:7-42, the compliance with theserecommendations amongst practicing gastroenterologists remains poor(see, Abrams J A, et al., Clin Gastroenterol Hepatol 2009). Indeedcompliance decreases with increasing BE segment length leading toincreasing rates of missed dysplasia. Other challenges with dysplasiadetection in BE include the spotty distribution of dysplasia in BE (see,Cameron A J, et al., Am J Gastroenterol 1997; 92:586-91) which leads tosampling error, poor inter-observer agreement amongst pathologists whilegrading dysplasia and the relatively poor sensitivity of currentsurveillance strategies in detecting prevalent dysplasia or carcinoma(see, Sharma P, et al., Gastroenterology 2004; 127:310-30). The utilityof advanced imaging techniques in the community remains unclear withonly a third of practicing gastroenterologists reporting use routinelyin BE surveillance (see, Singh M, et al., Gastrointestinal endoscopy2013; 78:689-95). Recently a sponge on a string device has been studiedin BE screening (see, Kadri S R, et al., Bmj 2010; 341:c4372). Thisdevice consists of a polyurethane foam sponge compressed in a gelatincapsule, attached to a string. The capsule is swallowed by the patient.The gelatin shell of the capsule dissolves in the gastric fluidreleasing the foam device as a sphere which is then pulled out with theattached string, providing brushing/cytology samples of the proximalstomach and esophagus. Biomarker studies can then be performed on thesesamples to detect BE. Two large multicenter studies have been performedin the United Kingdom with such a device using trefoil factor 3 (aprotein specific to BE epithelium) detected on immunohistochemistry as aBE marker, demonstrating the feasibility, safety and accuracy of thisapproach (see, Kadri S R, et al., Bmj 2010; 341:c4372; Ross-Innes C S,et al., PLoS medicine 2015; 12:e1001780). The sensitivity andspecificity of this marker in the detection of BE has been reported tobe 73% and 94% for BE segments of >1 cm in circumferential length.Additionally this capsule sponge device has been used safely in a studyconducted at Mayo Clinic Rochester in subjects with eosinophilicesophagitis (see, Katzka D A, et al., Clinical gastroenterology andhepatology: the official clinical practice journal of the AmericanGastroenterological Association 2015; 13:77-83 e2). Methylated DNAmarkers specific to BE epithelium (with and without dysplasia) have beendescribed (see, Kaz A M, et al., Cancer letters 2014; 342:193-9;Ahlquist D A, et al., Clinical gastroenterology and hepatology: theofficial clinical practice journal of the American GastroenterologicalAssociation 2012; 10:272-7 e1).

Provided herein is technology for esophageal disorder screening andparticularly, but not exclusively, to methods, compositions, and relateduses for detecting the presence of esophageal disorders (e.g., Barrett'sesophagus, Barrett's esophageal dysplasia, etc.). In addition, thetechnology provides methods, compositions and related uses fordistinguishing between Barrett's esophagus and Barrett's esophagealdysplasia, and between Barrett's esophageal low-grade dysplasia,Barrett's esophageal high-grade dysplasia, and esophageal adenocarcinomawithin samples obtained through whole esophageal swabbing or brushing oruse of a sponge capsule device.

As the technology is described herein, the section headings used are fororganizational purposes only and are not to be construed as limiting thesubject matter in any way.

In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein, a “nucleic acid” or “nucleic acid molecule” generallyrefers to any ribonucleic acid or deoxyribonucleic acid, which may beunmodified or modified DNA or RNA. “Nucleic acids” include, withoutlimitation, single- and double-stranded nucleic acids. As used herein,the term “nucleic acid” also includes DNA as described above thatcontains one or more modified bases. Thus, DNA with a backbone modifiedfor stability or for other reasons is a “nucleic acid”. The term“nucleic acid” as it is used herein embraces such chemically,enzymatically, or metabolically modified forms of nucleic acids, as wellas the chemical forms of DNA characteristic of viruses and cells,including for example, simple and complex cells.

The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or“nucleic acid” refer to a molecule having two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. Typical deoxyribonucleotides for DNA are thymine, adenine,cytosine, and guanine. Typical ribonucleotides for RNA are uracil,adenine, cytosine, and guanine.

As used herein, the terms “locus” or “region” of a nucleic acid refer toa subregion of a nucleic acid, e.g., a gene on a chromosome, a singlenucleotide, a CpG island, etc.

The terms “complementary” and “complementarity” refer to nucleotides(e.g., 1 nucleotide) or polynucleotides (e.g., a sequence ofnucleotides) related by the base-pairing rules. For example, thesequence 5′-A-G-T-3′ is complementary to the sequence 3′-T-C-A-5′.Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands effects theefficiency and strength of hybridization between nucleic acid strands.This is of particular importance in amplification reactions and indetection methods that depend upon binding between nucleic acids.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises coding sequences necessary for the production of an RNA,or of a polypeptide or its precursor. A functional polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence as long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, etc.) of thepolypeptide are retained. The term “portion” when used in reference to agene refers to fragments of that gene. The fragments may range in sizefrom a few nucleotides to the entire gene sequence minus one nucleotide.Thus, “a nucleotide comprising at least a portion of a gene” maycomprise fragments of the gene or the entire gene.

The term “gene” also encompasses the coding regions of a structural geneand includes sequences located adjacent to the coding region on both the5′ and 3′ ends, e.g., for a distance of about 1 kb on either end, suchthat the gene corresponds to the length of the full-length mRNA (e.g.,comprising coding, regulatory, structural and other sequences). Thesequences that are located 5′ of the coding region and that are presenton the mRNA are referred to as 5′ non-translated or untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′non-translated or 3′ untranslated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. In some organisms (e.g.,eukaryotes), a genomic form or clone of a gene contains the codingregion interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ ends of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, posttranscriptionalcleavage, and polyadenylation.

The term “wild-type” when made in reference to a gene refers to a genethat has the characteristics of a gene isolated from a naturallyoccurring source. The term “wild-type” when made in reference to a geneproduct refers to a gene product that has the characteristics of a geneproduct isolated from a naturally occurring source. The term“naturally-occurring” as applied to an object refers to the fact that anobject can be found in nature. For example, a polypeptide orpolynucleotide sequence that is present in an organism (includingviruses) that can be isolated from a source in nature and which has notbeen intentionally modified by the hand of a person in the laboratory isnaturally-occurring. A wild-type gene is often that gene or allele thatis most frequently observed in a population and is thus arbitrarilydesignated the “normal” or “wild-type” form of the gene. In contrast,the term “modified” or “mutant” when made in reference to a gene or to agene product refers, respectively, to a gene or to a gene product thatdisplays modifications in sequence and/or functional properties (e.g.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

The term “allele” refers to a variation of a gene; the variationsinclude but are not limited to variants and mutants, polymorphic loci,and single nucleotide polymorphic loci, frameshift, and splicemutations. An allele may occur naturally in a population or it mightarise during the lifetime of any particular individual of thepopulation.

Thus, the terms “variant” and “mutant” when used in reference to anucleotide sequence refer to a nucleic acid sequence that differs by oneor more nucleotides from another, usually related, nucleotide acidsequence. A “variation” is a difference between two different nucleotidesequences; typically, one sequence is a reference sequence.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (e.g., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (e.g., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Amplification of nucleic acids generally refers to the production ofmultiple copies of a polynucleotide, or a portion of the polynucleotide,typically starting from a small amount of the polynucleotide (e.g., asingle polynucleotide molecule, 10 to 100 copies of a polynucleotidemolecule, which may or may not be exactly the same), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S.Pat. No. 5,494,810) are forms of amplification. Additional types ofamplification include, but are not limited to, allele-specific PCR (see,e.g., U.S. Pat. No. 5,639,611), assembly PCR (see, e.g., U.S. Pat. No.5,965,408), helicase-dependent amplification (see, e.g., U.S. Pat. No.7,662,594), Hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and5,338,671), intersequence-specfic PCR, inverse PCR (see, e.g., Triglia,et al et al. (1988) Nucleic Acids Res., 16:8186), ligation-mediated PCR(see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25:1854-1858(1997); U.S. Pat. No. 5,508,169), methylation-specific PCR (see, e.g.,Herman, et al., (1996) PNAS 93(13) 9821-9826), miniprimer PCR, multiplexligation-dependent probe amplification (see, e.g., Schouten, et al.,(2002) Nucleic Acids Research 30(12): e57), multiplex PCR (see, e.g.,Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156;Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al.,(2008) BMC Genetics 9:80), nested PCR, overlap-extension PCR (see, e.g.,Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367), realtime PCR (see, e.g., Higuchi, et al et al., (1992) Biotechnology10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030), reversetranscription PCR (see, e.g., Bustin, S. A. (2000) J. MolecularEndocrinology 25:169-193), solid phase PCR, thermal asymmetricinterlaced PCR, and Touchdown PCR (see, e.g., Don, et al., Nucleic AcidsResearch (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5)812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485).Polynucleotide amplification also can be accomplished using digital PCR(see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004,(1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41,(1999); International Patent Publication No. WO05023091A2; US PatentApplication Publication No. 20070202525).

The term “polymerase chain reaction” (“PCR”) refers to the method of K.B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (“PCR”). Because thedesired amplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified” and are “PCR products” or “amplicons.”

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Q-beta replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038 [1972]).Other nucleic acid will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlin et al,Nature, 228:227 [1970]). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace (1989) Genomics4:560). Finally, thermostable template-dependant DNA polymerases (e.g.,Taq and Pfu DNA polymerases), by virtue of their ability to function athigh temperature, are found to display high specificity for thesequences bounded and thus defined by the primers; the high temperatureresults in thermodynamic conditions that favor primer hybridization withthe target sequences and not hybridization with non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press [1989]).

As used herein, the term “nucleic acid detection assay” refers to anymethod of determining the nucleotide composition of a nucleic acid ofinterest. Nucleic acid detection assay include but are not limited to,DNA sequencing methods, probe hybridization methods, structure specificcleavage assays (e.g., the INVADER assay, Hologic, Inc.) and aredescribed, e.g., in U.S. Pat. Nos. 5,846,717, 5,985,557, 5,994,069,6,001,567, 6,090,543, and 6,872,816; Lyamichev et al., Nat. Biotech.,17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009/0253142); enzyme mismatch cleavage methods (e.g., Variagenics, U.S.Pat. Nos. 6,110,684, 5,958,692, 5,851,770); polymerase chain reaction;branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481,5,710,264, 5,124,246, and 5,624,802); rolling circle replication (e.g.,U.S. Pat. Nos. 6,210,884, 6,183,960 and 6,235,502); NASBA (e.g., U.S.Pat. No. 5,409,818); molecular beacon technology (e.g., U.S. Pat. No.6,150,097); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229,6,221,583, 6,013,170, and 6,063,573); cycling probe technology (e.g.,U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988); Dade Behring signalamplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677,5,914,230, 5,882,867, and 5,792,614); ligase chain reaction (e.g.,Barnay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwichhybridization methods (e.g., U.S. Pat. No. 5,288,609).

The term “amplifiable nucleic acid” refers to a nucleic acid that may beamplified by any amplification method. It is contemplated that“amplifiable nucleic acid” will usually comprise “sample template.”

The term “sample template” refers to nucleic acid originating from asample that is analyzed for the presence of “target” (defined below). Incontrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,that is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productthat is complementary to a nucleic acid strand is induced, (e.g., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer, and theuse of the method.

The term “probe” refers to an oligonucleotide (e.g., a sequence ofnucleotides), whether occurring naturally as in a purified restrictiondigest or produced synthetically, recombinantly, or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification, and isolation of particulargene sequences (e.g., a “capture probe”). It is contemplated that anyprobe used in the present invention may, in some embodiments, be labeledwith any “reporter molecule,” so that is detectable in any detectionsystem, including, but not limited to enzyme (e.g., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. It is not intended that the present invention belimited to any particular detection system or label.

As used herein, “methylation” refers to cytosine methylation atpositions C5 or N4 of cytosine, the N6 position of adenine, or othertypes of nucleic acid methylation. In vitro amplified DNA is usuallyunmethylated because typical in vitro DNA amplification methods do notretain the methylation pattern of the amplification template. However,“unmethylated DNA” or “methylated DNA” can also refer to amplified DNAwhose original template was unmethylated or methylated, respectively.

Accordingly, as used herein a “methylated nucleotide” or a “methylatednucleotide base” refers to the presence of a methyl moiety on anucleotide base, where the methyl moiety is not present in a recognizedtypical nucleotide base. For example, cytosine does not contain a methylmoiety on its pyrimidine ring, but 5-methylcytosine contains a methylmoiety at position 5 of its pyrimidine ring. Therefore, cytosine is nota methylated nucleotide and 5-methylcytosine is a methylated nucleotide.In another example, thymine contains a methyl moiety at position 5 ofits pyrimidine ring; however, for purposes herein, thymine is notconsidered a methylated nucleotide when present in DNA since thymine isa typical nucleotide base of DNA.

As used herein, a “methylated nucleic acid molecule” refers to a nucleicacid molecule that contains one or more methylated nucleotides.

As used herein, a “methylation state”, “methylation profile”, and“methylation status” of a nucleic acid molecule refers to the presenceof absence of one or more methylated nucleotide bases in the nucleicacid molecule. For example, a nucleic acid molecule containing amethylated cytosine is considered methylated (e.g., the methylationstate of the nucleic acid molecule is methylated). A nucleic acidmolecule that does not contain any methylated nucleotides is consideredunmethylated.

The methylation state of a particular nucleic acid sequence (e.g., agene marker or DNA region as described herein) can indicate themethylation state of every base in the sequence or can indicate themethylation state of a subset of the bases (e.g., of one or morecytosines) within the sequence, or can indicate information regardingregional methylation density within the sequence with or withoutproviding precise information of the locations within the sequence themethylation occurs.

The methylation state of a nucleotide locus in a nucleic acid moleculerefers to the presence or absence of a methylated nucleotide at aparticular locus in the nucleic acid molecule. For example, themethylation state of a cytosine at the 7th nucleotide in a nucleic acidmolecule is methylated when the nucleotide present at the 7th nucleotidein the nucleic acid molecule is 5-methylcytosine. Similarly, themethylation state of a cytosine at the 7th nucleotide in a nucleic acidmolecule is unmethylated when the nucleotide present at the 7thnucleotide in the nucleic acid molecule is cytosine (and not5-methylcytosine).

The methylation status can optionally be represented or indicated by a“methylation value” (e.g., representing a methylation frequency,fraction, ratio, percent, etc.) A methylation value can be generated,for example, by quantifying the amount of intact nucleic acid presentfollowing restriction digestion with a methylation dependent restrictionenzyme or by comparing amplification profiles after bisulfite reactionor by comparing sequences of bisulfite-treated and untreated nucleicacids. Accordingly, a value, e.g., a methylation value, represents themethylation status and can thus be used as a quantitative indicator ofmethylation status across multiple copies of a locus. This is ofparticular use when it is desirable to compare the methylation status ofa sequence in a sample to a threshold or reference value.

As used herein, “methylation frequency” or “methylation percent (%)”refer to the number of instances in which a molecule or locus ismethylated relative to the number of instances the molecule or locus isunmethylated.

As such, the methylation state describes the state of methylation of anucleic acid (e.g., a genomic sequence). In addition, the methylationstate refers to the characteristics of a nucleic acid segment at aparticular genomic locus relevant to methylation. Such characteristicsinclude, but are not limited to, whether any of the cytosine (C)residues within this DNA sequence are methylated, the location ofmethylated C residue(s), the frequency or percentage of methylated Cthroughout any particular region of a nucleic acid, and allelicdifferences in methylation due to, e.g., difference in the origin of thealleles. The terms “methylation state”, “methylation profile”, and“methylation status” also refer to the relative concentration, absoluteconcentration, or pattern of methylated C or unmethylated C throughoutany particular region of a nucleic acid in a biological sample. Forexample, if the cytosine (C) residue(s) within a nucleic acid sequenceare methylated it may be referred to as “hypermethylated” or having“increased methylation”, whereas if the cytosine (C) residue(s) within aDNA sequence are not methylated it may be referred to as“hypomethylated” or having “decreased methylation”. Likewise, if thecytosine (C) residue(s) within a nucleic acid sequence are methylated ascompared to another nucleic acid sequence (e.g., from a different regionor from a different individual, etc.) that sequence is consideredhypermethylated or having increased methylation compared to the othernucleic acid sequence. Alternatively, if the cytosine (C) residue(s)within a DNA sequence are not methylated as compared to another nucleicacid sequence (e.g., from a different region or from a differentindividual, etc.) that sequence is considered hypomethylated or havingdecreased methylation compared to the other nucleic acid sequence.Additionally, the term “methylation pattern” as used herein refers tothe collective sites of methylated and unmethylated nucleotides over aregion of a nucleic acid. Two nucleic acids may have the same or similarmethylation frequency or methylation percent but have differentmethylation patterns when the number of methylated and unmethylatednucleotides are the same or similar throughout the region but thelocations of methylated and unmethylated nucleotides are different.Sequences are said to be “differentially methylated” or as having a“difference in methylation” or having a “different methylation state”when they differ in the extent (e.g., one has increased or decreasedmethylation relative to the other), frequency, or pattern ofmethylation. The term “differential methylation” refers to a differencein the level or pattern of nucleic acid methylation in a cancer positivesample as compared with the level or pattern of nucleic acid methylationin a cancer negative sample. It may also refer to the difference inlevels or patterns between patients that have recurrence of cancer aftersurgery versus patients who not have recurrence. Differentialmethylation and specific levels or patterns of DNA methylation areprognostic and predictive biomarkers, e.g., once the correct cut-off orpredictive characteristics have been defined.

Methylation state frequency can be used to describe a population ofindividuals or a sample from a single individual. For example, anucleotide locus having a methylation state frequency of 50% ismethylated in 50% of instances and unmethylated in 50% of instances.Such a frequency can be used, for example, to describe the degree towhich a nucleotide locus or nucleic acid region is methylated in apopulation of individuals or a collection of nucleic acids. Thus, whenmethylation in a first population or pool of nucleic acid molecules isdifferent from methylation in a second population or pool of nucleicacid molecules, the methylation state frequency of the first populationor pool will be different from the methylation state frequency of thesecond population or pool. Such a frequency also can be used, forexample, to describe the degree to which a nucleotide locus or nucleicacid region is methylated in a single individual. For example, such afrequency can be used to describe the degree to which a group of cellsfrom a tissue sample are methylated or unmethylated at a nucleotidelocus or nucleic acid region.

As used herein a “nucleotide locus” refers to the location of anucleotide in a nucleic acid molecule. A nucleotide locus of amethylated nucleotide refers to the location of a methylated nucleotidein a nucleic acid molecule.

Typically, methylation of human DNA occurs on a dinucleotide sequenceincluding an adjacent guanine and cytosine where the cytosine is located5′ of the guanine (also termed CpG dinucleotide sequences). Mostcytosines within the CpG dinucleotides are methylated in the humangenome, however some remain unmethylated in specific CpG dinucleotiderich genomic regions, known as CpG islands (see, e.g, Antequera et al.(1990) Cell 62: 503-514).

As used herein, a “CpG island” refers to a G:C-rich region of genomicDNA containing an increased number of CpG dinucleotides relative tototal genomic DNA. A CpG island can be at least 100, 200, or more basepairs in length, where the G:C content of the region is at least 50% andthe ratio of observed CpG frequency over expected frequency is 0.6; insome instances, a CpG island can be at least 500 base pairs in length,where the G:C content of the region is at least 55%) and the ratio ofobserved CpG frequency over expected frequency is 0.65. The observed CpGfrequency over expected frequency can be calculated according to themethod provided in Gardiner-Garden et al (1987) J. Mol. Biol. 196:261-281. For example, the observed CpG frequency over expected frequencycan be calculated according to the formula R=(A×B)/(C×D), where R is theratio of observed CpG frequency over expected frequency, A is the numberof CpG dinucleotides in an analyzed sequence, B is the total number ofnucleotides in the analyzed sequence, C is the total number of Cnucleotides in the analyzed sequence, and D is the total number of Gnucleotides in the analyzed sequence. Methylation state is typicallydetermined in CpG islands, e.g., at promoter regions. It will beappreciated though that other sequences in the human genome are prone toDNA methylation such as CpA and CpT (see, e.g., Ramsahoye (2000) Proc.Natl. Acad. Sci. USA 97: 5237-5242; Salmon and Kaye (1970) Biochim.Biophys. Acta. 204: 340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14: 4353-4367; Woodcock (1987)Biochem. Biophys. Res. Commun. 145: 888-894).

As used herein, a reagent that modifies a nucleotide of the nucleic acidmolecule as a function of the methylation state of the nucleic acidmolecule, or a methylation-specific reagent, refers to a compound orcomposition or other agent that can change the nucleotide sequence of anucleic acid molecule in a manner that reflects the methylation state ofthe nucleic acid molecule. Methods of treating a nucleic acid moleculewith such a reagent can include contacting the nucleic acid moleculewith the reagent, coupled with additional steps, if desired, toaccomplish the desired change of nucleotide sequence. Such a change inthe nucleic acid molecule's nucleotide sequence can result in a nucleicacid molecule in which each methylated nucleotide is modified to adifferent nucleotide. Such a change in the nucleic acid nucleotidesequence can result in a nucleic acid molecule in which eachunmethylated nucleotide is modified to a different nucleotide. Such achange in the nucleic acid nucleotide sequence can result in a nucleicacid molecule in which each of a selected nucleotide which isunmethylated (e.g., each unmethylated cytosine) is modified to adifferent nucleotide. Use of such a reagent to change the nucleic acidnucleotide sequence can result in a nucleic acid molecule in which eachnucleotide that is a methylated nucleotide (e.g., each methylatedcytosine) is modified to a different nucleotide. As used herein, use ofa reagent that modifies a selected nucleotide refers to a reagent thatmodifies one nucleotide of the four typically occurring nucleotides in anucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A forRNA), such that the reagent modifies the one nucleotide withoutmodifying the other three nucleotides. In one exemplary embodiment, sucha reagent modifies an unmethylated selected nucleotide to produce adifferent nucleotide. In another exemplary embodiment, such a reagentcan deaminate unmethylated cytosine nucleotides. An exemplary reagent isbisulfite.

As used herein, the term “bisulfite reagent” refers to a reagentcomprising in some embodiments bisulfite, disulfite, hydrogen sulfite,or combinations thereof to distinguish between methylated andunmethylated cytidines, e.g., in CpG dinucleotide sequences.

The term “methylation assay” refers to any assay for determining themethylation state of one or more CpG dinucleotide sequences within asequence of a nucleic acid.

The term “MS AP-PCR” (Methylation-Sensitive Arbitrarily-PrimedPolymerase Chain Reaction) refers to the art-recognized technology thatallows for a global scan of the genome using CG-rich primers to focus onthe regions most likely to contain CpG dinucleotides, and described byGonzalgo et al. (1997) Cancer Research 57: 594-599.

The term “MethyLight™” refers to the art-recognized fluorescence-basedreal-time PCR technique described by Eads et al. (1999) Cancer Res. 59:2302-2306.

The term “HeavyMethyl™” refers to an assay wherein methylation specificblocking probes (also referred to herein as blockers) covering CpGpositions between, or covered by, the amplification primers enablemethylation-specific selective amplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay refers to a HeavyMethyl™MethyLight™ assay, which is a variation of the MethyLight™ assay,wherein the MethyLight™ assay is combined with methylation specificblocking probes covering CpG positions between the amplificationprimers.

The term “Ms-SNuPE” (Methylation-sensitive Single Nucleotide PrimerExtension) refers to the art-recognized assay described by Gonzalgo &Jones (1997) Nucleic Acids Res. 25: 2529-2531.

The term “MSP” (Methylation-specific PCR) refers to the art-recognizedmethylation assay described by Herman et al. (1996) Proc. Natl. Acad.Sci. USA 93: 9821-9826, and by U.S. Pat. No. 5,786,146.

The term “COBRA” (Combined Bisulfite Restriction Analysis) refers to theart-recognized methylation assay described by Xiong & Laird (1997)Nucleic Acids Res. 25: 2532-2534.

The term “MCA” (Methylated CpG Island Amplification) refers to themethylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12, and in WO 00/26401A1.

As used herein, a “selected nucleotide” refers to one nucleotide of thefour typically occurring nucleotides in a nucleic acid molecule (C, G,T, and A for DNA and C, G, U, and A for RNA), and can include methylatedderivatives of the typically occurring nucleotides (e.g., when C is theselected nucleotide, both methylated and unmethylated C are includedwithin the meaning of a selected nucleotide), whereas a methylatedselected nucleotide refers specifically to a methylated typicallyoccurring nucleotide and an unmethylated selected nucleotides refersspecifically to an unmethylated typically occurring nucleotide.

The terms “methylation-specific restriction enzyme” or“methylation-sensitive restriction enzyme” refers to an enzyme thatselectively digests a nucleic acid dependent on the methylation state ofits recognition site. In the case of a restriction enzyme thatspecifically cuts if the recognition site is not methylated or ishemimethylated, the cut will not take place or will take place with asignificantly reduced efficiency if the recognition site is methylated.In the case of a restriction enzyme that specifically cuts if therecognition site is methylated, the cut will not take place or will takeplace with a significantly reduced efficiency if the recognition site isnot methylated. Preferred are methylation-specific restriction enzymes,the recognition sequence of which contains a CG dinucleotide (forinstance a recognition sequence such as CGCG or CCCGGG). Furtherpreferred for some embodiments are restriction enzymes that do not cutif the cytosine in this dinucleotide is methylated at the carbon atomC5.

As used herein, a “different nucleotide” refers to a nucleotide that ischemically different from a selected nucleotide, typically such that thedifferent nucleotide has Watson-Crick base-pairing properties thatdiffer from the selected nucleotide, whereby the typically occurringnucleotide that is complementary to the selected nucleotide is not thesame as the typically occurring nucleotide that is complementary to thedifferent nucleotide. For example, when C is the selected nucleotide, Uor T can be the different nucleotide, which is exemplified by thecomplementarity of C to G and the complementarity of U or T to A. Asused herein, a nucleotide that is complementary to the selectednucleotide or that is complementary to the different nucleotide refersto a nucleotide that base-pairs, under high stringency conditions, withthe selected nucleotide or different nucleotide with higher affinitythan the complementary nucleotide's base-paring with three of the fourtypically occurring nucleotides. An example of complementarity isWatson-Crick base pairing in DNA (e.g., A-T and C-G) and RNA (e.g., A-Uand C-G). Thus, for example, G base-pairs, under high stringencyconditions, with higher affinity to C than G base-pairs to G, A, or Tand, therefore, when C is the selected nucleotide, G is a nucleotidecomplementary to the selected nucleotide.

As used herein, the “sensitivity” of a given marker refers to thepercentage of samples that report a DNA methylation value above athreshold value that distinguishes between neoplastic and non-neoplasticsamples. In some embodiments, a positive is defined as ahistology-confirmed neoplasia that reports a DNA methylation value abovea threshold value (e.g., the range associated with disease), and a falsenegative is defined as a histology-confirmed neoplasia that reports aDNA methylation value below the threshold value (e.g., the rangeassociated with no disease). The value of sensitivity, therefore,reflects the probability that a DNA methylation measurement for a givenmarker obtained from a known diseased sample will be in the range ofdisease-associated measurements. As defined here, the clinical relevanceof the calculated sensitivity value represents an estimation of theprobability that a given marker would detect the presence of a clinicalcondition when applied to a subject with that condition.

As used herein, the “specificity” of a given marker refers to thepercentage of non-neoplastic samples that report a DNA methylation valuebelow a threshold value that distinguishes between neoplastic andnon-neoplastic samples. In some embodiments, a negative is defined as ahistology-confirmed non-neoplastic sample that reports a DNA methylationvalue below the threshold value (e.g., the range associated with nodisease) and a false positive is defined as a histology-confirmednon-neoplastic sample that reports a DNA methylation value above thethreshold value (e.g., the range associated with disease). The value ofspecificity, therefore, reflects the probability that a DNA methylationmeasurement for a given marker obtained from a known non-neoplasticsample will be in the range of non-disease associated measurements. Asdefined here, the clinical relevance of the calculated specificity valuerepresents an estimation of the probability that a given marker woulddetect the absence of a clinical condition when applied to a patientwithout that condition.

The term “AUC” as used herein is an abbreviation for the “area under acurve”. In particular it refers to the area under a Receiver OperatingCharacteristic (ROC) curve. The ROC curve is a plot of the true positiverate against the false positive rate for the different possible cutpoints of a diagnostic test. It shows the trade-off between sensitivityand specificity depending on the selected cut point (any increase insensitivity will be accompanied by a decrease in specificity). The areaunder an ROC curve (AUC) is a measure for the accuracy of a diagnostictest (the larger the area the better; the optimum is 1; a random testwould have a ROC curve lying on the diagonal with an area of 0.5; forreference: J. P. Egan. (1975) Signal Detection Theory and ROC Analysis,Academic Press, New York).

As used herein, the term “neoplasm” refers to “an abnormal mass oftissue, the growth of which exceeds and is uncoordinated with that ofthe normal tissues” See, e.g., Willis R A, “The Spread of Tumors in theHuman Body”, London, Butterworth & Co, 1952.

As used herein, the term “adenoma” refers to a benign tumor of glandularorigin. Although these growths are benign, over time they may progressto become malignant.

The term “pre-cancerous” or “pre-neoplastic” and equivalents thereofrefer to any cellular proliferative disorder that is undergoingmalignant transformation.

A “site” or “region” of a neoplasm, adenoma, cancer, etc. is the tissue,organ, cell type, anatomical area, body part, etc. in a subject's bodywhere the neoplasm, adenoma, cancer, etc. is located.

As used herein, the term “esophageal disorder” refers to types ofdisorder associated with the esophagus and/or esophageal tissue.Examples of esophageal disorders include, but are not limited to,Barrett's esophagus (BE), Barrett's esophageal dysplasia (BED),Barrett's esophageal low-grade dysplasia (BE-LGD), Barrett's esophagealhigh-grade dysplasia (BE-HGD), and esophageal adenocarcinoma (EAC).

As used herein, a “diagnostic” test application includes the detectionor identification of a disease state or condition of a subject,determining the likelihood that a subject will contract a given diseaseor condition, determining the likelihood that a subject with a diseaseor condition will respond to therapy, determining the prognosis of asubject with a disease or condition (or its likely progression orregression), and determining the effect of a treatment on a subject witha disease or condition. For example, a diagnostic can be used fordetecting the presence or likelihood of a subject contracting a neoplasmor the likelihood that such a subject will respond favorably to acompound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

The term “marker”, as used herein, refers to a substance (e.g., anucleic acid or a region of a nucleic acid) that is able to diagnose adisorder (e.g., a non-cancerous disorder) (e.g., a cancerous disorder)by distinguishing disorder-associated cells (e.g., non-cancerous cellsassociated with the disorder) (e.g., cancerous cells associated with thedisorder) from normal cells, e.g., based its methylation state.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” refers to a nucleic acid sequence that isidentified and separated from at least one contaminant nucleic acid withwhich it is ordinarily associated in its natural source. Isolatednucleic acid is present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acids,such as DNA and RNA, are found in the state they exist in nature.Examples of non-isolated nucleic acids include: a given DNA sequence(e.g., a gene) found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, found in the cell as a mixture withnumerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a particular protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the protein,where the nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature. The isolated nucleic acid oroligonucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid or oligonucleotide is to be utilizedto express a protein, the oligonucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide may besingle-stranded), but may contain both the sense and anti-sense strands(i.e., the oligonucleotide may be double-stranded). An isolated nucleicacid may, after isolation from its natural or typical environment, by becombined with other nucleic acids or molecules. For example, an isolatednucleic acid may be present in a host cell in which into which it hasbeen placed, e.g., for heterologous expression.

The term “purified” refers to molecules, either nucleic acid or aminoacid sequences that are removed from their natural environment,isolated, or separated. An “isolated nucleic acid sequence” maytherefore be a purified nucleic acid sequence. “Substantially purified”molecules are at least 60% free, preferably at least 75% free, and morepreferably at least 90% free from other components with which they arenaturally associated. As used herein, the terms “purified” or “topurify” also refer to the removal of contaminants from a sample. Theremoval of contaminating proteins results in an increase in the percentof polypeptide or nucleic acid of interest in the sample. In anotherexample, recombinant polypeptides are expressed in plant, bacterial,yeast, or mammalian host cells and the polypeptides are purified by theremoval of host cell proteins; the percent of recombinant polypeptidesis thereby increased in the sample.

The term “composition comprising” a given polynucleotide sequence orpolypeptide refers broadly to any composition containing the givenpolynucleotide sequence or polypeptide. The composition may comprise anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS),and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

The term “sample” is used in its broadest sense. In one sense it canrefer to an animal cell or tissue. In another sense, it is meant toinclude a specimen or culture obtained from any source, as well asbiological and environmental samples. Biological samples may be obtainedfrom plants or animals (including humans) and encompass fluids, solids,tissues, and gases. Environmental samples include environmental materialsuch as surface matter, soil, water, and industrial samples. Theseexamples are not to be construed as limiting the sample types applicableto the present invention. In some embodiments, the sample includesesophageal tissue. In some embodiments, the sample includes esophagealtissue obtained through endoscopic brushing or nonendoscopic wholeesophageal brushing or swabbing using a tethered device (e.g. such as acapsule sponge, balloon, or other device).

As used herein, a “remote sample” as used in some contexts relates to asample indirectly collected from a site that is not the cell, tissue, ororgan source of the sample. For instance, when sample materialoriginating from the pancreas is assessed in a stool sample (e.g., notfrom a sample taken directly from a pancreas), the sample is a remotesample.

As used herein, the terms “patient” or “subject” refer to organisms tobe subject to various tests provided by the technology. The term“subject” includes animals, preferably mammals, including humans. In apreferred embodiment, the subject is a primate. In an even morepreferred embodiment, the subject is a human.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.As used herein, the term “fragmented kit” refers to delivery systemscomprising two or more separate containers that each contain asubportion of the total kit components.

The containers may be delivered to the intended recipient together orseparately. For example, a first container may contain an enzyme for usein an assay, while a second container contains oligonucleotides. Theterm “fragmented kit” is intended to encompass kits containing Analytespecific reagents (ASR's) regulated under section 520(e) of the FederalFood, Drug, and Cosmetic Act, but are not limited thereto. Indeed, anydelivery system comprising two or more separate containers that eachcontains a subportion of the total kit components are included in theterm “fragmented kit.” In contrast, a “combined kit” refers to adelivery system containing all of the components of a reaction assay ina single container (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

EMBODIMENTS OF THE TECHNOLOGY

Barrett's Esophagus is a precursor lesion for most esophagealadenocarcinomas which is a malignancy with rapidly rising incidence andpersistently poor outcomes. Early detection of esophageal adenocarcinomahas been shown to be associated with earlier stage and increasedsurvival. Early detection of Barrett's Esophagus may enable placement ofpatients into surveillance programs which may allow detection ofneoplastic progression at an earlier stage amenable to endoscopic orsurgical therapy with improved outcomes. Screening for Barrett'sEsophagus and esophageal adenocarcinoma has been hampered by the lack ofa widely applicable tool, as well as the lack of a biomarker which canbe combined with a screening tool. Acceptability and feasibility ofscreening by endoscopic and novel non-endoscopic methods has beendemonstrated in the population. Non-endoscopic screening methods, suchas by swallowed cytology brush or stool DNA testing, offer potentialcost-effective alternatives to endoscopy for identification of Barrett'sEsophagus in the general population. More recently, it has also shownthat several aberrantly methylated genes could serve as highlydiscriminant markers for Barrett's Esophagus. Indeed, a study performedon archived frozen esophageal biopsies in patients with and withoutBarrett's revealed that a panel of tumor-associated genes waspotentially useful to discriminate between Barrett's Esophagus andsquamous mucosa. (see, e.g., Yang Wu, et al, DDW Abstract 2011).

Dysplasia is known to be distributed in a patchy manner in Barrett'sesophagus, leading to “sampling error” on routine endoscopicsurveillance as performed by four quadrant biopsies. It is known thatconventional endoscopic surveillance with biopsies samples less than 10%of the BE segment. Compliance of endoscopists with conventionalsurveillance is known to be poor. While newer endoscopic techniques havebeen shown to improve the yield of dysplasia detection in studiesperformed in tertiary care centers, their applicability in the communityremains uncertain. Methods which sample a larger mucosal surface area,such as swabbing or brushing, are likely to increase the yield ofdysplasia and neoplasia, particularly if combined with molecular markersof dysplasia/neoplasia. This may ultimately allow non-biopsy (viaswabbing or brushing) or non-endoscopic surveillance of BE subjects withpotential substantial cost savings.

Accordingly, provided herein is technology for esophageal disorderscreening and particularly, but not exclusively, to methods,compositions, and related uses for detecting the presence of esophagealdisorders (e.g., Barrett's esophagus, Barrett's esophageal dysplasia,etc.). In addition, the technology provides methods, compositions andrelated uses for distinguishing between Barrett's esophagus andBarrett's esophageal dysplasia, and between Barrett's esophageallow-grade dysplasia, Barrett's esophageal high-grade dysplasia, andesophageal adenocarcinoma within samples obtained through endoscopicbrushing or nonendoscopic whole esophageal brushing or swabbing using atethered device (e.g. such as a capsule sponge, balloon, or otherdevice).

Indeed, experiments conducted during the course of developing thistechnology compared the methylation state of DNA markers from esophagealtissue of subjects having Barrett's esophagus to the methylation stateof the same DNA markers from control subjects (e.g., normal tissue forthe respective tissue type), and to the methylation state of the sameDNA markers from subjects having Barrett's esophagus dysplasia (see,Examples 1-4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Tables 1, 7 and/or 8) capable ofclassifying Barrett's esophagus (BE) versus control (e.g., normal tissuefor the respective tissue type) within esophageal tissue (see, Examples1, 2 and 5).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Tables 2, 3, 5, and/or 6)capable of classifying BE versus Barrett's esophagus related dyspasia(BED) within esophageal tissue (see, Examples 1, 3 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of predictingBarrett's esophagus related low-grade dyspasia (BE-LGD), Barrett'sesophagus related dyspasia high-grade dysplasia (BE-HGD), and esophagealadenocarcinoma (EAC) within samples obtained through whole esophagealswabbing or brushing (see, Example 1 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of classifyingBE versus BED within samples obtained through whole esophageal swabbingor brushing (see, Example 1 and 4).

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

The methods comprise determining the methylation status of at least onemethylation marker in a biological sample isolated from a subject,wherein a change in the methylation state of the marker is indicative ofthe presence, or class of an esophageal disorder (e.g., BE, BED, BE-LGD,BE-HGD, EAC). Particular embodiments relate to markers comprising adifferentially methylated region (DMR, e.g., DMR 1-229, see Tables 1, 2,3, 5, 6, 7, 8) that are used for diagnosis (e.g., screening) ofesophageal disorders (e.g., BE, BED, BE-LGD, BE-HGD, EAC), includingearly detection during, for example, pre-cancerous stages of disease(e.g., BE versus BED).

The markers of the present technology are particularly efficient indetecting or distinguishing between esophageal disorders (e.g., BE, BED,BE-LGD, BE-HGD, EAC), thereby providing improved means for the earlydetection, classification, and treatment of said disorders.

In addition to embodiments wherein the methylation analysis of at leastone marker, a region of a marker, or a base of a marker comprising a DMR(e.g., DMR 1-229 from Tables 1, 2, 3, 5, 6, 7, and/or 8) provided hereinand listed in Tables 1, 2, 3, 5, 6, and/or 7 is analyzed, the technologyalso provides panels of markers comprising at least one marker, regionof a marker, or base of a marker comprising a DMR with utility for thedetection of esophageal disorders (e.g., BE, BED, BE-LGD, BE-HGD, EAC),in esophageal tissue.

Some embodiments of the technology are based upon the analysis of theCpG methylation status of at least one marker, region of a marker, orbase of a marker comprising a DMR.

In some embodiments, the present technology provides for the use of thebisulfite technique in combination with one or more methylation assaysto determine the methylation status of CpG dinucleotide sequences withinat least one marker comprising a DMR (e.g., as provided in Tables 1, 2,3, 5, 6, 7, 8 (e.g., DMR 1-229)). Genomic CpG dinucleotides can bemethylated or unmethylated (alternatively known as up- anddown-methylated respectively). However the methods of the presentinvention are suitable for the analysis of biological samples of aheterogeneous nature, e.g., a low concentration of tumor cells, orbiological materials therefrom, within a background of a remote sample(e.g., blood, organ effluent, or stool). Accordingly, when analyzing themethylation status of a CpG position within such a sample one may use aquantitative assay for determining the level (e.g., percent, fraction,ratio, proportion, or degree) of methylation at a particular CpGposition.

According to the present technology, determination of the methylationstatus of CpG dinucleotide sequences in markers comprising a DMR hasutility both in the diagnosis and characterization of esophagealdisorders (e.g., BE, BED, BE-LGD, BE-HGD, EAC).

Combinations of Markers

In some embodiments, the technology relates to assessing the methylationstate of combinations of markers comprising two or more DMRs from Tables1, 2, 3, 5, 6, 7, 8 (e.g., two or more DMRs from DMR Nos. 1-194). Insome embodiments, assessing the methylation state of more than onemarker increases the specificity and/or sensitivity of a screen ordiagnostic for identifying an esophageal disorder (e.g., BE, BED,BE-LGD, BE-HGD, EAC) in a subject.

Various cancers are predicted by various combinations of markers, e.g.,as identified by statistical techniques related to specificity andsensitivity of prediction. The technology provides methods foridentifying predictive combinations and validated predictivecombinations for some cancers.

In some embodiments, combinations of markers (e.g., comprising a DMR)predict the site of a neoplasm.

For example, markers and/or panels of markers were identified (e.g., achromosomal region having an annotation provided in Tables 1, 7 and/or8) capable of classifying Barrett's esophagus (BE) versus control (e.g.,normal tissue for the respective tissue type) within esophageal tissue(see, Examples 1, 2 and 5).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Tables 2, 3, 5, and/or 6)capable of classifying BE versus Barrett's esophagus related dyspasia(BED) within esophageal tissue (see, Examples 1, 3 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of predictingBarrett's esophagus related low-grade dyspasia (BE-LGD), Barrett'sesophagus related dyspasia high-grade dysplasia (BE-HGD), and esophagealadenocarcinoma (EAC) within samples obtained through whole esophagealswabbing or brushing (see, Examples 1 and 4).

Markers and/or panels of markers were identified (e.g., a chromosomalregion having an annotation provided in Table 5) capable of classifyingBE versus BED within samples obtained through whole esophageal swabbingor brushing (see, Examples 1 and 4).

Methods for Assaying Methylation State

The most frequently used method for analyzing a nucleic acid for thepresence of 5-methylcytosine is based upon the bisulfite methoddescribed by Frommer, et al. for the detection of 5-methylcytosines inDNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-31) orvariations thereof. The bisulfite method of mapping 5-methylcytosines isbased on the observation that cytosine, but not 5-methylcytosine, reactswith hydrogen sulfite ion (also known as bisulfite). The reaction isusually performed according to the following steps: first, cytosinereacts with hydrogen sulfite to form a sulfonated cytosine. Next,spontaneous deamination of the sulfonated reaction intermediate resultsin a sulfonated uracil. Finally, the sulfonated uricil is desulfonatedunder alkaline conditions to form uracil. Detection is possible becauseuracil forms base pairs with adenine (thus behaving like thymine),whereas 5-methylcytosine base pairs with guanine (thus behaving likecytosine). This makes the discrimination of methylated cytosines fromnon-methylated cytosines possible by, e.g., bisulfite genomic sequencing(Grigg G, & Clark S, Bioessays (1994) 16: 431-36; Grigg G, DNA Seq.(1996) 6: 189-98) or methylation-specific PCR (MSP) as is disclosed,e.g., in U.S. Pat. No. 5,786,146.

Some conventional technologies are related to methods comprisingenclosing the DNA to be analyzed in an agarose matrix, therebypreventing the diffusion and renaturation of the DNA (bisulfite onlyreacts with single-stranded DNA), and replacing precipitation andpurification steps with a fast dialysis (Olek A, et al. (1996) “Amodified and improved method for bisulfite based cytosine methylationanalysis” Nucleic Acids Res. 24: 5064-6). It is thus possible to analyzeindividual cells for methylation status, illustrating the utility andsensitivity of the method. An overview of conventional methods fordetecting 5-methylcytosine is provided by Rein, T., et al. (1998)Nucleic Acids Res. 26: 2255. The bisulfite technique typically involvesamplifying short, specific fragments of a known nucleic acid subsequentto a bisulfite treatment, then either assaying the product by sequencing(Olek & Walter (1997) Nat. Genet. 17: 275-6) or a primer extensionreaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25: 2529-31; WO95/00669; U.S. Pat. No. 6,251,594) to analyze individual cytosinepositions. Some methods use enzymatic digestion (Xiong & Laird (1997)Nucleic Acids Res. 25: 2532-4). Detection by hybridization has also beendescribed in the art (Olek et al., WO 99/28498). Additionally, use ofthe bisulfite technique for methylation detection with respect toindividual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6; Zeschnigk et al. (1997) Hum Mol Genet. 6: 387-95; Feil et al.(1994) Nucleic Acids Res. 22: 695; Martin et al. (1995) Gene 157: 261-4;WO 9746705; WO 9515373).

Various methylation assay procedures are known in the art and can beused in conjunction with bisulfite treatment according to the presenttechnology. These assays allow for determination of the methylationstate of one or a plurality of CpG dinucleotides (e.g., CpG islands)within a nucleic acid sequence. Such assays involve, among othertechniques, sequencing of bisulfite-treated nucleic acid, PCR (forsequence-specific amplification), Southern blot analysis, and use ofmethylation-sensitive restriction enzymes.

For example, genomic sequencing has been simplified for analysis ofmethylation patterns and 5-methylcytosine distributions by usingbisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA89: 1827-1831). Additionally, restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA finds use in assessingmethylation state, e.g., as described by Sadri & Hornsby (1997) Nucl.Acids Res. 24: 5058-5059 or as embodied in the method known as COBRA(Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) NucleicAcids Res. 25: 2532-2534).

COBRA™ analysis is a quantitative methylation assay useful fordetermining DNA methylation levels at specific loci in small amounts ofgenomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).Briefly, restriction enzyme digestion is used to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Methylation-dependent sequence differences arefirst introduced into the genomic DNA by standard bisulfite treatmentaccording to the procedure described by Frommer et al. (Proc. Natl.Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfiteconverted DNA is then performed using primers specific for the CpGislands of interest, followed by restriction endonuclease digestion, gelelectrophoresis, and detection using specific, labeled hybridizationprobes. Methylation levels in the original DNA sample are represented bythe relative amounts of digested and undigested PCR product in alinearly quantitative fashion across a wide spectrum of DNA methylationlevels. In addition, this technique can be reliably applied to DNAobtained from microdissected paraffin-embedded tissue samples.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit)for COBRA™ analysis may include, but are not limited to: PCR primers forspecific loci (e.g., specific genes, markers, DMR, regions of genes,regions of markers, bisulfite treated DNA sequence, CpG island, etc.);restriction enzyme and appropriate buffer; gene-hybridizationoligonucleotide; control hybridization oligonucleotide; kinase labelingkit for oligonucleotide probe; and labeled nucleotides. Additionally,bisulfite conversion reagents may include: DNA denaturation buffer;sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation,ultrafiltration, affinity column); desulfonation buffer; and DNArecovery components.

Preferably, assays such as “MethyLight™” (a fluorescence-based real-timePCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPETM(Methylation-sensitive Single Nucleotide Primer Extension) reactions(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997),methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci.USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpGisland amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12,1999) are used alone or in combination with one or more of thesemethods.

The “HeavyMethyl™” assay, technique is a quantitative method forassessing methylation differences based on methylation-specificamplification of bisulfite-treated DNA. Methylation-specific blockingprobes (“blockers”) covering CpG positions between, or covered by, theamplification primers enable methylation-specific selectiveamplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay refers to a HeavyMethyl™MethyLight™ assay, which is a variation of the MethyLight™ assay,wherein the MethyLight™ assay is combined with methylation specificblocking probes covering CpG positions between the amplificationprimers. The HeavyMethyl™ assay may also be used in combination withmethylation specific amplification primers.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for HeavyMethyl™ analysis may include, but are not limited to: PCRprimers for specific loci (e.g., specific genes, markers, DMR, regionsof genes, regions of markers, bisulfite treated DNA sequence, CpGisland, or bisulfite treated DNA sequence or CpG island, etc.); blockingoligonucleotides; optimized PCR buffers and deoxynucleotides; and Taqpolymerase.

MSP (methylation-specific PCR) allows for assessing the methylationstatus of virtually any group of CpG sites within a CpG island,independent of the use of methylation-sensitive restriction enzymes(Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat.No. 5,786,146). Briefly, DNA is modified by sodium bisulfite, whichconverts unmethylated, but not methylated cytosines, to uracil, and theproducts are subsequently amplified with primers specific for methylatedversus unmethylated DNA. MSP requires only small quantities of DNA, issensitive to 0.1% methylated alleles of a given CpG island locus, andcan be performed on DNA extracted from paraffin-embedded samples.Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for specific loci (e.g., specific genes,markers, DMR, regions of genes, regions of markers, bisulfite treatedDNA sequence, CpG island, etc.); optimized PCR buffers anddeoxynucleotides, and specific probes.

The MethyLight™ assay is a high-throughput quantitative methylationassay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®)that requires no further manipulations after the PCR step (Eads et al.,Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process beginswith a mixed sample of genomic DNA that is converted, in a sodiumbisulfite reaction, to a mixed pool of methylation-dependent sequencedifferences according to standard procedures (the bisulfite processconverts unmethylated cytosine residues to uracil). Fluorescence-basedPCR is then performed in a “biased” reaction, e.g., with PCR primersthat overlap known CpG dinucleotides. Sequence discrimination occursboth at the level of the amplification process and at the level of thefluorescence detection process.

The MethyLight™ assay is used as a quantitative test for methylationpatterns in a nucleic acid, e.g., a genomic DNA sample, wherein sequencediscrimination occurs at the level of probe hybridization. In aquantitative version, the PCR reaction provides for a methylationspecific amplification in the presence of a fluorescent probe thatoverlaps a particular putative methylation site. An unbiased control forthe amount of input DNA is provided by a reaction in which neither theprimers, nor the probe, overlie any CpG dinucleotides. Alternatively, aqualitative test for genomic methylation is achieved by probing thebiased PCR pool with either control oligonucleotides that do not coverknown methylation sites (e.g., a fluorescence-based version of theHeavyMethyl™ and MSP techniques) or with oligonucleotides coveringpotential methylation sites.

The MethyLight™ process is used with any suitable probe (e.g. a“TaqMan®” probe, a Lightcycler® probe, etc.) For example, in someapplications double-stranded genomic DNA is treated with sodiumbisulfite and subjected to one of two sets of PCR reactions usingTaqMan® probes, e.g., with MSP primers and/or HeavyMethyl blockeroligonucleotides and a TaqMan® probe. The TaqMan® probe is dual-labeledwith fluorescent “reporter” and “quencher” molecules and is designed tobe specific for a relatively high GC content region so that it melts atabout a 10° C. higher temperature in the PCR cycle than the forward orreverse primers. This allows the TaqMan® probe to remain fullyhybridized during the PCR annealing/extension step. As the Taqpolymerase enzymatically synthesizes a new strand during PCR, it willeventually reach the annealed TaqMan® probe. The Taq polymerase 5′ to 3′endonuclease activity will then displace the TaqMan® probe by digestingit to release the fluorescent reporter molecule for quantitativedetection of its now unquenched signal using a real-time fluorescentdetection system.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for MethyLight™ analysis may include, but are not limited to: PCRprimers for specific loci (e.g., specific genes, markers, DMR, regionsof genes, regions of markers, bisulfite treated DNA sequence, CpGisland, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

The QM™ (quantitative methylation) assay is an alternative quantitativetest for methylation patterns in genomic DNA samples, wherein sequencediscrimination occurs at the level of probe hybridization. In thisquantitative version, the PCR reaction provides for unbiasedamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe, overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing the biased PCR poolwith either control oligonucleotides that do not cover known methylationsites (a fluorescence-based version of the HeavyMethyl™ and MSPtechniques) or with oligonucleotides covering potential methylationsites.

The QM™ process can be used with any suitable probe, e.g., “TaqMan®”probes, Lightcycler® probes, in the amplification process. For example,double-stranded genomic DNA is treated with sodium bisulfite andsubjected to unbiased primers and the TaqMan® probe. The TaqMan® probeis dual-labeled with fluorescent “reporter” and “quencher” molecules,and is designed to be specific for a relatively high GC content regionso that it melts out at about a 10° C. higher temperature in the PCRcycle than the forward or reverse primers. This allows the TaqMan® probeto remain fully hybridized during the PCR annealing/extension step. Asthe Taq polymerase enzymatically synthesizes a new strand during PCR, itwill eventually reach the annealed TaqMan® probe. The Taq polymerase 5′to 3′ endonuclease activity will then displace the TaqMan® probe bydigesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system. Typical reagents (e.g., as might be foundin a typical QM™-based kit) for QM™ analysis may include, but are notlimited to: PCR primers for specific loci (e.g., specific genes,markers, DMR, regions of genes, regions of markers, bisulfite treatedDNA sequence, CpG island, etc.); TaqMan® or Lightcycler® probes;optimized PCR buffers and deoxynucleotides; and Taq polymerase.

The Ms-SNuPETM technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly,genomic DNA is reacted with sodium bisulfite to convert unmethylatedcytosine to uracil while leaving 5-methylcytosine unchanged.Amplification of the desired target sequence is then performed using PCRprimers specific for bisulfite-converted DNA, and the resulting productis isolated and used as a template for methylation analysis at the CpGsite of interest. Small amounts of DNA can be analyzed (e.g.,microdissected pathology sections) and it avoids utilization ofrestriction enzymes for determining the methylation status at CpG sites.

Typical reagents (e.g., as might be found in a typical Ms-SNuPETM-basedkit) for Ms-SNuPETM analysis may include, but are not limited to: PCRprimers for specific loci (e.g., specific genes, markers, DMR, regionsof genes, regions of markers, bisulfite treated DNA sequence, CpGisland, etc.); optimized PCR buffers and deoxynucleotides; gelextraction kit; positive control primers; Ms-SNuPETM primers forspecific loci; reaction buffer (for the Ms-SNuPE reaction); and labelednucleotides. Additionally, bisulfite conversion reagents may include:DNA denaturation buffer; sulfonation buffer; DNA recovery reagents orkit (e.g., precipitation, ultrafiltration, affinity column);desulfonation buffer; and DNA recovery components.

Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfitetreatment of nucleic acid to convert all unmethylated cytosines touracil, followed by restriction enzyme digestion (e.g., by an enzymethat recognizes a site including a CG sequence such as MspI) andcomplete sequencing of fragments after coupling to an adapter ligand.The choice of restriction enzyme enriches the fragments for CpG denseregions, reducing the number of redundant sequences that may map tomultiple gene positions during analysis. As such, RRBS reduces thecomplexity of the nucleic acid sample by selecting a subset (e.g., bysize selection using preparative gel electrophoresis) of restrictionfragments for sequencing. As opposed to whole-genome bisulfitesequencing, every fragment produced by the restriction enzyme digestioncontains DNA methylation information for at least one CpG dinucleotide.As such, RRBS enriches the sample for promoters, CpG islands, and othergenomic features with a high frequency of restriction enzyme cut sitesin these regions and thus provides an assay to assess the methylationstate of one or more genomic loci.

A typical protocol for RRBS comprises the steps of digesting a nucleicacid sample with a restriction enzyme such as MspI, filling in overhangsand A-tailing, ligating adaptors, bisulfite conversion, and PCR. See,e.g., et al. (2005) “Genome-scale DNA methylation mapping of clinicalsamples at single-nucleotide resolution” Nat Methods 7: 133-6; Meissneret al. (2005) “Reduced representation bisulfite sequencing forcomparative high-resolution DNA methylation analysis” Nucleic Acids Res.33: 5868-77.

In some embodiments, a quantitative allele-specific real-time target andsignal amplification (QUARTS) assay is used to evaluate methylationstate. Three reactions sequentially occur in each QuARTS assay,including amplification (reaction 1) and target probe cleavage (reaction2) in the primary reaction; and FRET cleavage and fluorescent signalgeneration (reaction 3) in the secondary reaction. When target nucleicacid is amplified with specific primers, a specific detection probe witha flap sequence loosely binds to the amplicon. The presence of thespecific invasive oligonucleotide at the target binding site causescleavase to release the flap sequence by cutting between the detectionprobe and the flap sequence. The flap sequence is complementary to anonhairpin portion of a corresponding FRET cassette. Accordingly, theflap sequence functions as an invasive oligonucleotide on the FRETcassette and effects a cleavage between the FRET cassette fluorophoreand a quencher, which produces a fluorescent signal. The cleavagereaction can cut multiple probes per target and thus release multiplefluorophore per flap, providing exponential signal amplification. QuARTScan detect multiple targets in a single reaction well by using FRETcassettes with different dyes. See, e.g., in Zou et al. (2010)“Sensitive quantification of methylated markers with a novel methylationspecific technology” Clin Chem 56: A199; U.S. patent application Ser.Nos. 12/946,737, 12/946,745, 12/946,752, and 61/548,639.

The term “bisulfite reagent” refers to a reagent comprising bisulfite,disulfite, hydrogen sulfite, or combinations thereof, useful asdisclosed herein to distinguish between methylated and unmethylated CpGdinucleotide sequences. Methods of said treatment are known in the art(e.g., PCT/EP2004/011715). It is preferred that the bisulfite treatmentis conducted in the presence of denaturing solvents such as but notlimited to n-alkylenglycol or diethylene glycol dimethyl ether (DME), orin the presence of dioxane or dioxane derivatives. In some embodimentsthe denaturing solvents are used in concentrations between 1% and 35%(v/v). In some embodiments, the bisulfite reaction is carried out in thepresence of scavengers such as but not limited to chromane derivatives,e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane 2-carboxylic acid ortrihydroxybenzone acid and derivates thereof, e.g., Gallic acid (see:PCT/EP2004/011715). The bisulfite conversion is preferably carried outat a reaction temperature between 30° C. and 70° C., whereby thetemperature is increased to over 85° C. for short times during thereaction (see: PCT/EP2004/011715). The bisulfite treated DNA ispreferably purified prior to the quantification. This may be conductedby any means known in the art, such as but not limited toultrafiltration, e.g., by means of Microcon™ columns (manufactured byMillipore™). The purification is carried out according to a modifiedmanufacturer's protocol (see, e.g., PCT/EP2004/011715).

In some embodiments, fragments of the treated DNA are amplified usingsets of primer oligonucleotides according to the present invention(e.g., see Tables 4 and 9) and an amplification enzyme. Theamplification of several DNA segments can be carried out simultaneouslyin one and the same reaction vessel. Typically, the amplification iscarried out using a polymerase chain reaction (PCR). Amplicons aretypically 100 to 2000 base pairs in length.

In another embodiment of the method, the methylation status of CpGpositions within or near a marker comprising a DMR (e.g., DMR 1-229 asprovided in Tables 1, 2, 3, 5, 6, 7, 8) may be detected by use ofmethylation-specific primer oligonucleotides. This technique (MSP) hasbeen described in U.S. Pat. No. 6,265,171 to Herman. The use ofmethylation status specific primers for the amplification of bisulfitetreated DNA allows the differentiation between methylated andunmethylated nucleic acids. MSP primer pairs contain at least one primerthat hybridizes to a bisulfite treated CpG dinucleotide. Therefore, thesequence of said primers comprises at least one CpG dinucleotide. MSPprimers specific for non-methylated DNA contain a “T” at the position ofthe C position in the CpG.

The fragments obtained by means of the amplification can carry adirectly or indirectly detectable label. In some embodiments, the labelsare fluorescent labels, radionuclides, or detachable molecule fragmentshaving a typical mass that can be detected in a mass spectrometer. Wheresaid labels are mass labels, some embodiments provide that the labeledamplicons have a single positive or negative net charge, allowing forbetter delectability in the mass spectrometer. The detection may becarried out and visualized by means of, e.g., matrix assisted laserdesorption/ionization mass spectrometry (MALDI) or using electron spraymass spectrometry (ESI).

Methods for isolating DNA suitable for these assay technologies areknown in the art. In particular, some embodiments comprise isolation ofnucleic acids as described in U.S. patent application Ser. No.13/470,251 (“Isolation of Nucleic Acids”).

Methods

In some embodiments the technology, methods are provided that comprisethe following steps:

-   -   1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated        from a body fluids such as a stool sample, a blood sample, or a        tissue sample (e.g., esophageal tissue)) obtained from a subject        with at least one reagent or series of reagents that        distinguishes between methylated and non-methylated CpG        dinucleotides within at least one marker comprising a DMR (e.g.,        DMR 1-78 as provided in Table 1, DMR 21, 188-193 as provided in        Table 7, DMR 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108,        122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164,        166, 171, 173, 175, 178, 179, 181, 185, 187, 193-229 as provided        in Table 8) and    -   2) detecting a lack of Barrett's esophagus (e.g., afforded with        a sensitivity of greater than or equal to 80% and a specificity        of greater than or equal to 80%).

In some embodiments the technology, methods are provided that comprisethe following steps:

-   -   1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated        from a body fluids such as a stool sample, a blood sample, or a        tissue sample (e.g., esophageal tissue)) obtained from a subject        with at least one reagent or series of reagents that        distinguishes between methylated and non-methylated CpG        dinucleotides within at least one marker comprising a DMR (e.g.,        DMR 1-78 as provided in Table 1, DMR 21, 188-193 as provided in        Table 7, DMR 2-4, 6, 7, 14, 30, 77, 80, 82-86, 88, 90-102, 108,        122, 135, 136, 141, 142, 144, 146, 148-149, 152, 154, 156, 164,        166, 171, 173, 175, 178, 179, 181, 185, 187, 193-229 as provided        in Table 8) and    -   2) detecting a presence of Barrett's esophagus (e.g., afforded        with a sensitivity of greater than or equal to 80% and a        specificity of greater than or equal to 80%).

In some embodiments the technology, methods are provided that comprisethe following steps:

-   -   1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated        from a body fluids such as a stool sample, a blood sample, or a        tissue sample (e.g., esophageal tissue)) obtained from a subject        with at least one reagent or series of reagents that        distinguishes between methylated and non-methylated CpG        dinucleotides within at least one marker comprising a DMR (e.g.,        DMR No. 3, 5, 30, 33, 43, 58, 77 and 79-128 as provided in Table        2, DMR No. 77, 27, 193, 90, 92, 101 and 129-134 as provided in        Table 3, DMR No. 77, 90 and 135 as provided in Table 5, DMR No.        136-187 as provided in Table 6) and    -   2) classifying Barrett's esophagus or Barrett's esophageal        dysplasia (e.g., afforded with a sensitivity of greater than or        equal to 80% and a specificity of greater than or equal to 80%).

In some embodiments the technology, methods are provided that comprisethe following steps:

-   -   1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated        from esophageal tissue (e.g., esophageal tissue obtained through        whole esophageal swabbing or brushing)) obtained from a subject        with at least one reagent or series of reagents that        distinguishes between methylated and non-methylated CpG        dinucleotides within at least one marker comprising a DMR (e.g.,        DMR No. 77, 90 and 135 as provided in Table 5) and    -   2) classifying Barrett's esophagus or Barrett's esophageal        dysplasia (e.g., afforded with a sensitivity of greater than or        equal to 80% and a specificity of greater than or equal to 80%).

In some embodiments the technology, methods are provided that comprisethe following steps:

-   -   1) contacting a nucleic acid (e.g., genomic DNA, e.g., isolated        from esophageal tissue (e.g., esophageal tissue obtained through        whole esophageal swabbing or brushing)) obtained from a subject        with at least one reagent or series of reagents that        distinguishes between methylated and non-methylated CpG        dinucleotides within at least one marker comprising a DMR (e.g.,        DMR No. 77, 90 and 135 as provided in Table 5) and    -   2) classifying Barrett's esophageal low-grade dysplasia,        Barrett's esophageal high-grade dysplasia, or esophageal        adenocarcinoma (e.g., afforded with a sensitivity of greater        than or equal to 80% and a specificity of greater than or equal        to 80%).        Preferably, the sensitivity is from about 70% to about 100%, or        from about 80% to about 90%, or from about 80% to about 85%.        Preferably, the specificity is from about 70% to about 100%, or        from about 80% to about 90%, or from about 80% to about 85%.

Genomic DNA may be isolated by any means, including the use ofcommercially available kits. Briefly, wherein the DNA of interest isencapsulated in by a cellular membrane the biological sample must bedisrupted and lysed by enzymatic, chemical or mechanical means. The DNAsolution may then be cleared of proteins and other contaminants, e.g.,by digestion with proteinase K. The genomic DNA is then recovered fromthe solution. This may be carried out by means of a variety of methodsincluding salting out, organic extraction, or binding of the DNA to asolid phase support. The choice of method will be affected by severalfactors including time, expense, and required quantity of DNA. Allclinical sample types comprising neoplastic matter or pre-neoplasticmatter are suitable for use in the present method, e.g., cell lines,histological slides, biopsies, paraffin-embedded tissue, body fluids,stool, colonic effluent, urine, blood plasma, blood serum, whole blood,isolated blood cells, cells isolated from the blood, and combinationsthereof.

In some embodiments wherein the sample includes esophageal tissue, thesample is obtained through endoscopic techniques.

In some embodiments wherein the sample includes esophageal tissue, thesample is obtained through endoscopic brushing or nonendoscopic wholeesophageal brushing or swabbing using a tethered device (e.g. such as acapsule sponge, balloon, or other device).

The technology is not limited in the methods used to prepare the samplesand provide a nucleic acid for testing. For example, in someembodiments, a DNA is isolated from a stool sample or from blood or froma plasma sample using direct gene capture, e.g., as detailed in U.S.Pat. Appl. Ser. No. 61/485,386 or by a related method.

The genomic DNA sample is then treated with at least one reagent, orseries of reagents, that distinguishes between methylated andnon-methylated CpG dinucleotides within at least one marker comprising aDMR (e.g., DMR 1-229, e.g., as provided by Tables 1, 2, 3, 5, 6, 7, 8).

In some embodiments, the reagent converts cytosine bases which areunmethylated at the 5′-position to uracil, thymine, or another basewhich is dissimilar to cytosine in terms of hybridization behavior.However in some embodiments, the reagent may be a methylation sensitiverestriction enzyme.

In some embodiments, the genomic DNA sample is treated in such a mannerthat cytosine bases that are unmethylated at the 5′ position areconverted to uracil, thymine, or another base that is dissimilar tocytosine in terms of hybridization behavior. In some embodiments, thistreatment is carried out with bisulfate (hydrogen sulfite, disulfite)followed byt alkaline hydrolysis.

The treated nucleic acid is then analyzed to determine the methylationstate of the target gene sequences (at least one gene, genomic sequence,or nucleotide from a marker comprising a DMR, e.g., at least one DMRchosen from DMR 1-229, e.g., as provided in Tables 1, 2, 3, 5, 6, 7, 8).The method of analysis may be selected from those known in the art,including those listed herein, e.g., QuARTS and MSP as described herein.

The technology relates to the analysis of any sample associated with anesophageal disorder (e.g., BE, BED, BE-LGD, BE-HGD, EAC). For example,in some embodiments the sample comprises a tissue and/or biologicalfluid obtained from a patient. In some embodiments, the sample comprisesesophageal tissue. In some embodiments, the sample comprises esophagealtissue obtained through whole esophageal swabbing or brushing. In someembodiments, the sample comprises a secretion. In some embodiments, thesample comprises blood, serum, plasma, gastric secretions, pancreaticjuice, a gastrointestinal biopsy sample, microdissected cells from anesophageal biopsy, esophageal cells sloughed into the gastrointestinallumen, and/or esophageal cells recovered from stool. In someembodiments, the subject is human. These samples may originate from theupper gastrointestinal tract, the lower gastrointestinal tract, orcomprise cells, tissues, and/or secretions from both the uppergastrointestinal tract and the lower gastrointestinal tract. The samplemay include cells, secretions, or tissues from the liver, bile ducts,pancreas, stomach, colon, rectum, esophagus, small intestine, appendix,duodenum, polyps, gall bladder, anus, and/or peritoneum. In someembodiments, the sample comprises cellular fluid, ascites, urine, feces,pancreatic fluid, fluid obtained during endoscopy, blood, mucus, orsaliva. In some embodiments, the sample is a stool sample.

Such samples can be obtained by any number of means known in the art,such as will be apparent to the skilled person. For instance, urine andfecal samples are easily attainable, while blood, ascites, serum, orpancreatic fluid samples can be obtained parenterally by using a needleand syringe, for instance. Cell free or substantially cell free samplescan be obtained by subjecting the sample to various techniques known tothose of skill in the art which include, but are not limited to,centrifugation and filtration. Although it is generally preferred thatno invasive techniques are used to obtain the sample, it still may bepreferable to obtain samples such as tissue homogenates, tissuesections, and biopsy specimens. In some embodiments, the sample isobtained through esophageal swabbing or brushing or use of a spongecapsule device.

In some embodiments, the technology relates to a method for treating apatient (e.g., a patient with BE, BED, BE-LGD, BE-HGD, and/or EAC), themethod comprising determining the methylation state of one or more DMRas provided herein and administering a treatment to the patient based onthe results of determining the methylation state. The treatment may beadministration of a pharmaceutical compound, a vaccine, performing asurgery, imaging the patient, performing another test. Preferably, saiduse is in a method of clinical screening, a method of prognosisassessment, a method of monitoring the results of therapy, a method toidentify patients most likely to respond to a particular therapeutictreatment, a method of imaging a patient or subject, and a method fordrug screening and development.

In some embodiments of the technology, a method for diagnosing anesophageal disorder (e.g., BE, BED, BE-LGD, BE-HGD, EAC) in a subject isprovided. The terms “diagnosing” and “diagnosis” as used herein refer tomethods by which the skilled artisan can estimate and even determinewhether or not a subject is suffering from a given disease or conditionor may develop a given disease or condition in the future. The skilledartisan often makes a diagnosis on the basis of one or more diagnosticindicators, such as for example a biomarker (e.g., a DMR as disclosedherein), the methylation state of which is indicative of the presence,severity, or absence of the condition.

Along with diagnosis, clinical cancer prognosis (e.g., for BED, BE-LGD,BE-HGD, EAC) relates to determining the aggressiveness of the cancer andthe likelihood of tumor recurrence to plan the most effective therapy.If a more accurate prognosis can be made or even a potential risk fordeveloping the cancer can be assessed, appropriate therapy, and in someinstances less severe therapy for the patient can be chosen. Assessment(e.g., determining methylation state) of cancer biomarkers is useful toseparate subjects with good prognosis and/or low risk of developingcancer who will need no therapy or limited therapy from those morelikely to develop cancer or suffer a recurrence of cancer who mightbenefit from more intensive treatments.

As such, “making a diagnosis” or “diagnosing”, as used herein, isfurther inclusive of making determining a risk of developing cancer ordetermining a prognosis, which can provide for predicting a clinicaloutcome (with or without medical treatment), selecting an appropriatetreatment (or whether treatment would be effective), or monitoring acurrent treatment and potentially changing the treatment, based on themeasure of the diagnostic biomarkers (e.g., DMR) disclosed herein.Further, in some embodiments of the presently disclosed subject matter,multiple determinations of the biomarkers over time can be made tofacilitate diagnosis and/or prognosis. A temporal change in thebiomarker can be used to predict a clinical outcome, monitor theprogression of esophageal disorder, and/or monitor the efficacy ofappropriate therapies directed against the cancer. In such an embodimentfor example, one might expect to see a change in the methylation stateof one or more biomarkers (e.g., DMR) disclosed herein (and potentiallyone or more additional biomarker(s), if monitored) in a biologicalsample over time during the course of an effective therapy.

The presently disclosed subject matter further provides in someembodiments a method for determining whether to initiate or continueprophylaxis or treatment of an esophageal disorder (e.g., BE, BED,BE-LGD, BE-HGD, EAC) in a subject. In some embodiments, the methodcomprises providing a series of biological samples over a time periodfrom the subject; analyzing the series of biological samples todetermine a methylation state of at least one biomarker disclosed hereinin each of the biological samples; and comparing any measurable changein the methylation states of one or more of the biomarkers in each ofthe biological samples. Any changes in the methylation states ofbiomarkers over the time period can be used to predict risk ofdeveloping the esophageal disorder, predict clinical outcome, determinewhether to initiate or continue the prophylaxis or therapy of thecancer, and whether a current therapy is effectively treating theesophageal disorder. For example, a first time point can be selectedprior to initiation of a treatment and a second time point can beselected at some time after initiation of the treatment. Methylationstates can be measured in each of the samples taken from different timepoints and qualitative and/or quantitative differences noted. A changein the methylation states of the biomarker levels from the differentsamples can be correlated with esophageal disorder risk, prognosis,determining treatment efficacy, and/or progression of the esophagealdisorder in the subject.

In preferred embodiments, the methods and compositions of the inventionare for treatment or diagnosis of disease at an early stage, forexample, before symptoms of the disease appear. In some embodiments, themethods and compositions of the invention are for treatment or diagnosisof disease at a clinical stage.

As noted, in some embodiments, multiple determinations of one or morediagnostic or prognostic biomarkers can be made, and a temporal changein the marker can be used to determine a diagnosis or prognosis. Forexample, a diagnostic marker can be determined at an initial time, andagain at a second time. In such embodiments, an increase in the markerfrom the initial time to the second time can be diagnostic of aparticular type or severity of the esophageal disorder, or a givenprognosis. Likewise, a decrease in the marker from the initial time tothe second time can be indicative of a particular type or severity of anesophageal disorder, or a given prognosis. Furthermore, the degree ofchange of one or more markers can be related to the severity of theesophageal disorder and future adverse events. The skilled artisan willunderstand that, while in certain embodiments comparative measurementscan be made of the same biomarker at multiple time points, one can alsomeasure a given biomarker at one time point, and a second biomarker at asecond time point, and a comparison of these markers can providediagnostic information.

As used herein, the phrase “determining the prognosis” refers to methodsby which the skilled artisan can predict the course or outcome of acondition in a subject. The term “prognosis” does not refer to theability to predict the course or outcome of a condition with 100%accuracy, or even that a given course or outcome is predictably more orless likely to occur based on the methylation state of a biomarker(e.g., a DMR). Instead, the skilled artisan will understand that theterm “prognosis” refers to an increased probability that a certaincourse or outcome will occur; that is, that a course or outcome is morelikely to occur in a subject exhibiting a given condition, when comparedto those individuals not exhibiting the condition. For example, inindividuals not exhibiting the condition (e.g., having a normalmethylation state of one or more DMR), the chance of a given outcome(e.g., suffering from an esophageal disorder) may be very low.

In some embodiments, a statistical analysis associates a prognosticindicator with a predisposition to an adverse outcome. For example, insome embodiments, a methylation state different from that in a normalcontrol sample obtained from a patient who does not have an esophagealdisorder can signal that a subject is more likely to suffer from anesophageal disorder than subjects with a level that is more similar tothe methylation state in the control sample, as determined by a level ofstatistical significance. Additionally, a change in methylation statefrom a baseline (e.g., “normal”) level can be reflective of subjectprognosis, and the degree of change in methylation state can be relatedto the severity of adverse events. Statistical significance is oftendetermined by comparing two or more populations and determining aconfidence interval and/or a p value (see, e.g., Dowdy and Wearden,Statistics for Research, John Wiley & Sons, New York, 1983). Exemplaryconfidence intervals of the present subject matter are 90%, 95%, 97.5%,98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p values are 0.1,0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

In other embodiments, a threshold degree of change in the methylationstate of a prognostic or diagnostic biomarker disclosed herein (e.g., aDMR) can be established, and the degree of change in the methylationstate of the biamarker in a biological sample is simply compared to thethreshold degree of change in the methylation state. A preferredthreshold change in the methylation state for biomarkers provided hereinis about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 50%, about 75%, about 100%, and about 150%. In yet otherembodiments, a “nomogram” can be established, by which a methylationstate of a prognostic or diagnostic indicator (biomarker or combinationof biomarkers) is directly related to an associated disposition towardsa given outcome. The skilled artisan is acquainted with the use of suchnomograms to relate two numeric values with the understanding that theuncertainty in this measurement is the same as the uncertainty in themarker concentration because individual sample measurements arereferenced, not population averages.

In some embodiments, a control sample is analyzed concurrently with thebiological sample, such that the results obtained from the biologicalsample can be compared to the results obtained from the control sample.Additionally, it is contemplated that standard curves can be provided,with which assay results for the biological sample may be compared. Suchstandard curves present methylation states of a biomarker as a functionof assay units, e.g., fluorescent signal intensity, if a fluorescentlabel is used. Using samples taken from multiple donors, standard curvescan be provided for control methylation states of the one or morebiomarkers in normal tissue, as well as for “at-risk” levels of the oneor more biomarkers in tissue taken from donors with metaplasia or fromdonors with an esophageal disorder (e.g., BE, BED, BE-LGD, BE-HGD, EAC).In certain embodiments of the method, a subject is identified as havingan esophageal disorder upon identifying an aberrant methylation state ofone or more DMR provided herein in a biological sample obtained from thesubject. In other embodiments of the method, the detection of anaberrant methylation state of one or more of such biomarkers in abiological sample obtained from the subject results in the subject beingidentified as having an esophageal disorder (e.g., BE, BED, BE-LGD,BE-HGD, EAC).

The analysis of markers can be carried out separately or simultaneouslywith additional markers within one test sample. For example, severalmarkers can be combined into one test for efficient processing of amultiple of samples and for potentially providing greater diagnosticand/or prognostic accuracy. In addition, one skilled in the art wouldrecognize the value of testing multiple samples (for example, atsuccessive time points) from the same subject. Such testing of serialsamples can allow the identification of changes in marker methylationstates over time. Changes in methylation state, as well as the absenceof change in methylation state, can provide useful information about thedisease status that includes, but is not limited to, identifying theapproximate time from onset of the event, the presence and amount ofsalvageable tissue, the appropriateness of drug therapies, theeffectiveness of various therapies, and identification of the subject'soutcome, including risk of future events.

The analysis of biomarkers can be carried out in a variety of physicalformats. For example, the use of microtiter plates or automation can beused to facilitate the processing of large numbers of test samples.Alternatively, single sample formats could be developed to facilitateimmediate treatment and diagnosis in a timely fashion, for example, inambulatory transport or emergency room settings.

In some embodiments, the subject is diagnosed as having an esophagealdisorder (e.g., BE, BED, BE-LGD, BE-HGD, EAC) if, when compared to acontrol methylation state, there is a measurable difference in themethylation state of at least one biomarker in the sample. Conversely,when no change in methylation state is identified in the biologicalsample, the subject can be identified as not having an esophagealdisorder, not being at risk for the esophageal disorder, or as having alow risk of the esophageal disorder. In this regard, subjects having theesophageal disorder or risk thereof can be differentiated from subjectshaving low to substantially no esophageal disorder or risk thereof.Those subjects having a risk of developing an esophageal disorder can beplaced on a more intensive and/or regular screening schedule, includingendoscopic surveillance. On the other hand, those subjects having low tosubstantially no risk may avoid being subjected to an endoscopy oresophageal brushing, until such time as a future screening, for example,a screening conducted in accordance with the present technology,indicates that a risk of esophageal disorder has appeared in thosesubjects.

As mentioned above, depending on the embodiment of the method of thepresent technology, detecting a change in methylation state of the oneor more biomarkers can be a qualitative determination or it can be aquantitative determination. As such, the step of diagnosing a subject ashaving, or at risk of developing, an esophageal disorder indicates thatcertain threshold measurements are made, e.g., the methylation state ofthe one or more biomarkers in the biological sample varies from apredetermined control methylation state. In some embodiments of themethod, the control methylation state is any detectable methylationstate of the biomarker. In other embodiments of the method where acontrol sample is tested concurrently with the biological sample, thepredetermined methylation state is the methylation state in the controlsample. In other embodiments of the method, the predeterminedmethylation state is based upon and/or identified by a standard curve.In other embodiments of the method, the predetermined methylation stateis a specifically state or range of state. As such, the predeterminedmethylation state can be chosen, within acceptable limits that will beapparent to those skilled in the art, based in part on the embodiment ofthe method being practiced and the desired specificity, etc.

Further with respect to diagnostic methods, a preferred subject is avertebrate subject. A preferred vertebrate is warm-blooded; a preferredwarm-blooded vertebrate is a mammal. A preferred mammal is mostpreferably a human. As used herein, the term “subject” includes bothhuman and animal subjects. Thus, veterinary therapeutic uses areprovided herein. As such, the present technology provides for thediagnosis of mammals such as humans, as well as those mammals ofimportance due to being endangered, such as Siberian tigers; of economicimportance, such as animals raised on farms for consumption by humans;and/or animals of social importance to humans, such as animals kept aspets or in zoos. Examples of such animals include but are not limitedto: carnivores such as cats and dogs; swine, including pigs, hogs, andwild boars; ruminants and/or ungulates such as cattle, oxen, sheep,giraffes, deer, goats, bison, and camels; and horses. Thus, alsoprovided is the diagnosis and treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), and the like. The presently-disclosed subject matterfurther includes a system for diagnosing an esophageal disorder (e.g.,BE, BED, BE-LGD, BE-HGD, EAC) in a subject. The system can be provided,for example, as a commercial kit that can be used to screen for a riskof esophageal disorder or diagnose an esophageal disorder in a subjectfrom whom a biological sample has been collected. An exemplary systemprovided in accordance with the present technology includes assessingthe methylation state of a DMR as provided in Tables 1, 2, 3, 5, 6, 7and/or 8.

Examples Example I

Molecular markers may aid in detection of Barrett's esophagus (BE) andsurveillance of BE-related dysplasia (BED) by either endoscopic ornon-endoscopic methods. Experiments were conducted to (1) identify andvalidate novel methylated DNA markers for BE dysplasia, (2) test thefeasibility of candidate markers for detection of BE dysplasia fromwhole-esophageal brushings.

Using whole methylome bisulfate sequencing on DNA from BE tissues withno dysplasia, low grade dysplasia (BE-LGD), high grade dysplasia(BE-HGD) or adenocarcinoma (EAC) (18 specimens per group), candidatemarkers were identified to separate BE from normal tissue, and toseparate BED from BE without dysplasia.

Tables 1 and 7 provide DMR information including chromosome number, geneannotation, and DMR start/stop position for such markers identified toseparate BE from normal tissue (see, Example II for materials/methodsutilized in generating Tables 1 and 7).

Tables 2 and 6 provide DMR information including chromosome number, geneannotation, and DMR start/stop position for such markers identified toseparate BED from BE without dysplasia (see, Example III formaterials/methods utilized in generating Tables 2 and 6). Top candidatemarkers were validated by methylation-specific PCR assay in independenttissues including BE without dysplasia, BE-LGD, and BE-HGD (30-36specimens per group).

Consenting BE subjects scheduled for endoscopic BE surveillance orendoscopic assessment of BE related cancers underwent whole esophagealbrushings using a high capacity cytology brush (Hobbs Medical, StaffordSprings, Conn.) with circumferential sampling from the cardia throughthe full esophageal length (BE+squamous mucosa) to simulate a swallowedsponge-on-string device. Following DNA extraction and bisulfatetreatment, methylation on target genes was assayed bymethylation-specific PCR or quantitative allele-specific real-timetarget and signal amplification. Marker levels were normalized toβ-actin (marker for total human DNA). 12 aberrantly methylated geneswere identified that best discriminated BED with from BE withoutdysplasia (e.g. areas under ROC curve 0.86-0.97) (see, Table 3). These12 markers were DIO3, MAX20.218, CD1D, T-SPYLS, ZNF568, ST8SIA1, ELMO1,ELOVL2, BMP3, NDRG4, HUNK, and CDKN2A. Table 3 DMR provides informationincluding chromosome number, gene annotation, and DMR start/stopposition for such markers identified to separate BED from BE withoutdysplasia within esophageal samples obtained through whole esophagealbrushings. Table 4 provides forward primer and reverse primerinformation for the DMRs provided in Table 3. 39 subjects were studiedwith a median age of 69 (28-94) years, 74% were males, and median BElength was 4 (1-14) cm; 18 had no dysplasia and 21 had dysplasia (9 LGD,7 HGD, and 5 EAC (4 asymptomatic early stage). A 3 marker set (DIO3,MAX20.218, NDRG4) (see, Table 5) at 95% specificity detected 78% of LGD,71% of HGD, 100% of EAC and 81% of all dysplasia (see, FIG. 1). Table 5provides DMR information including chromosome number, gene annotation,and DMR start/stop position for such markers identified to distinguishbetween LGD, HGD and EAC.

TABLE 1 Information for DMRs distinguishing BE and normal tissue DMRChromosome Gene DMR Start DMR End No. No. Annotation Transcript PositionPosition 1 chr19 ZNF256 NM_005773 58459137 58459219 2 chr19 ZNF568NM_198539 37407197 37407284 3 chr5 IRX4 NM_016358 1883238 1883312 4 chr6RGS17 NM_012419 153451813 153451881 5 chr7 GLI3 NM_000168 4227686242277220 6 chr4 EPHA5 NM_182472; 66536122 66536220 NM_004439 7 chr10SFMBT2 NM_001029880; 7451771 7451869 NM_001018039 8 chr3 WNT5A NM_00339255522021 55522106 9 chr4 VEGFC NM_005429 177713309 177713364 10 chr5ZNF354C NM_014594 178487249 178487299 11 chr19 ZNF85 NM_003429; 2110604321106185 NR_034060 12 chr8 FAM150A NM_207413 53477546 53477636 13 chr1NTNG1 NM_014917; 107684356 107684482 NM_001113226; NM_001113228 14 chr19A1BG NM_130786 58859193 58859258 15 chr13 SPG20 NM_001142294; 3692093336921108 NM_015087; NM_001142296; NM_001142295 16 chr3 EPHA6NM_001080448 96533015 96533096 17 chr16 FOXF1 NM_001451 8654235586542441 18 chr2 MYT1L NM_015025 1821558 1821642 19 chr9 — — 9944925099449346 20 chr7 — — 84815089 84815157 21 chr19 ZNF682 NM_001077349;20149796 20149923 NM_033196 22 chr8 PREX2 NM_025170; 68864872 68864921NM_024870 23 chr1 WNT3A NM_033131 228195339 228195413 24 chr7 TFPI2NM_006528 93520157 93520217 25 chr1 EDARADD NM_145861; 236559238236559336 NM_080738 26 chr1 WNT3A NM_033131 228195101 228195175 27 chr16NDRG4 NM_020465; 58497251 58497332 NM_001130487; NM_022910 28 chr19 — —15090770 15090853 29 chr8 — — 49782979 49783039 30 chr12 DPY19L2NM_173812 64061896 64062007 31 chr14 — — 97685552 97685636 32 chr2EFEMP1 NM_004105; 56150932 56150987 NM_001039348; NM_001039349 33 chr17NGFR NM_002507 47574211 47574294 34 chr8 PREX2 NM_025170; 6886492768865051 NM_024870 35 chr2 PXDN NM_012293 1748578 1748660 36 chr8C8orf42 NM_175075 494156 494193 37 chr16 DKFZP434H168 NR_026889 5622844856228463 38 chr14 FLRT2 NM_013231 85998492 85998535 39 chr20 SOX18NM_018419 62680089 62680150 40 chr1 PIK3CD NM_005026 9711854 9711974 41chr13 NALCN NM_052867 102069229 102069258 42 chr15 ATP10A NM_02449026108587 26108685 43 chr10 GRID1 NM_017551 88125585 88125655 44 chr18NOL4 NM_003787; 31802599 31802655 NM_001198548; NM_001198546;NM_001198547; NR_036752 45 chr5 FSTL4 NM_015082 132946635 132946746 46chr16 DKFZP434H168 NR_026889 56228468 56228505 47 chr12 TBC1D30NM_015279 65218475 65218525 48 chr2 GAL3ST2 NM_022134 242742873242743049 49 chr12 — — 47225496 47225592 50 chr11 FOLH1 NM_001193472;49229987 49230073 NM_001193473; NM_004476; NM_001193471; NM_001014986 51chr14 FLJ43390 NR_015358 62584108 62584204 52 chr21 TIAM1 NM_00325332932297 32932372 53 chr4 SLIT2 NM_004787 20254997 20255028 54 chr6 — —28979210 28979409 55 chr2 — — 1554621 1554768 56 chr12 PTPRO NM_002848;15475654 15475697 NM_030667 57 chr4 HAND2 NM_021973 174451394 17445143958 chr4 — — 180980619 180980711 59 chr10 PPAPDC1A NM_001030059 122216135122216312 60 chr17 FMNL1 NM_005892 43298763 43298872 61 chr4 FAT4NM_024582 126237876 126237908 62 chr1 PRRX1 NM_006902; 170633637170633683 NM_022716 63 chr5 SLC27A6 NM_014031; 128301108 128301233NM_001017372 64 chr18 TCF4 NM_001083962; 53257019 53257106 NM_003199 65chr14 FLRT2 NM_013231 85997993 85998139 66 chr20 SLC32A1 NM_08055237353717 37353740 67 chr8 KCNB2 NM_004770 73450042 73450129 68 chr7DPY19L2P4 NR_003551 89747980 89748001 69 chr17 — — 19483467 19483522 70chr10 GRID1 NM_017551 88125122 88125227 71 chr6 B3GAT2 NM_08074271666972 71667038 72 chr7 — — 42533077 42533175 73 chr19 ANKRD27NM_032139 33167174 33167250 74 chr4 GABRA2 NM_001114175; 4639239946392486 NM_000807 75 chr1 — — 39044435 39044453 76 chr3 CHL1 NM_006614238318 238401 77 chr14 DIO3 NM_001362 102026104 102026145 78 chr9IGFBPL1 NM_001007563 38424583 38424652

TABLE 2 Information for DMRs Distinguishing BE from BED DMR ChomosomeGene DMR Start DMR Stop No. No. Annotation Transcript Position Position79 chr10 — — 71267810 71267844 80 chr7 WNT2 NM_003391; 116964596116964659 NR_024047 81 chr7 ARPC1B NM_005720 98990762 98990837 82 chr19RGL3 NM_001035223; 11529371 11529430 NM_001161616 83 chr15 FEM1BNM_015322 68569729 68569799 84 chr15 ARNT2 NM_014862 80696170 8069617785 chr15 LARP6 NM_197958; 71146759 71146820 NM_018357 86 chr7 ZC3HAV1LNM_080660 138720915 138720957 87 chr2 CYBRD1 NM_024843; 172379904172379997 NM_001127383 88 chr15 Max.chr15.41877531.41877548 — 4187753141877548 89 chr7 GTF2IRD1 NM_005685; 73894929 73895008 NM_001199207;NM_016328 90 chr20 Max.chr20.2188420.2188480 — 2188420 2188480 91 chr18KLHL14 NM_020805 30351268 30351486 92 chr21 HUNK NM_014586 3324658033246650 93 chr19 LOC100131691 NR_027334 59073783 59073952 94 chr13Max.chr13.95620964.95621061 — 95620964 95621061 95 chr5Max.chr5.926920.927009 — 926920 927009 96 chr6 C6orf114 NM_03306913488436 13488530 97 chr8 ARHGEF10 NM_014629 1771362 1771477 98 chr20VSTM2L NM_080607 36531194 36531312 99 chr3 ACAD11 NM_032169 132378234132378296 100 chr12 WSB2 NM_018639 118500206 118500305 101 chr9 CDKN2ANM_000077; 21974710 21974763 NM_001195132; NM_058195; NM_058197 102 chr6Max.chr6.27064706.27064783 — 27064706 27064783 103 chr6 SGK1NM_001143676 134638972 134639020 104 chr6 SLC35B3 NM_015948; 84360748436140 NM_001142540; NM_001142541 105 chr1 PDE4DIP NM_022359; 145039649145039883 NM_001198832 106 chr3 SOX2OT NR_004053 181413970 181414052 107chr2 KLHL29 NM_052920 23609989 23610069 108 chr12 WIF1 NM_00719165514995 65515089 109 chr5 EBF1 NM_024007 158526068 158526167 110 chr11RDX NM_002906 110167594 110167690 111 chr6 LOC100526820 NR_037593163837485 163837640 5 chr7 GLI3 NM_000168 42276862 42277220 112 chr7 EN2NM_001427 155249880 155249949 113 chr10 ZNF365 NM_199450; 6413379464133834 NM_199451; NM_014951 114 chr12 — — 59990783 59990950 115 chr2 —— 238480870 238480950 116 chr19 RYR1 NM_001042723; 39055744 39055882NM_000540 117 chr3 PTPRG NM_002841 61549380 61549403 118 chr20 CYP24A1NM_001128915; 52790139 52790206 NM_000782 119 chr19 GDF15 NM_00486418499563 18499621 120 chr17 ULK2 NM_001142610; 19771310 19771382NM_014683 121 chr18 SETBP1 NM_015559; 42261225 42261288 NM_001130110 122chr7 DLX5 NM_005221 96653893 96653955 123 chr12 TRPV4 NM_021625110271304 110271388 77 chr14 DIO3 NM_001362 102026104 102026145 43 chr10GRID1 NM_017551 88125585 88125655 58 chr4 — — 180980619 180980711 124chr16 GPT2 NM_133443; 46963785 46963821 NM_001142466 125 chr10 PIP4K2ANM_005028 23003771 23003865 126 chr4 — — 184718393 184718464 127 chr14 —— 103726953 103727098 128 chr2 LOC91149 NR_026995 173600924 173601006 33chr17 NGFR NM_002507 47574211 47574294 3 chr5 IRX4 NM_016358 18832381883312 30 chr12 DPY19L2 NM_173812 64061896 64062007

TABLE 3 Information for DMRs Distinguishing BE from BED DMR ChromosomeGene DMR Start DMR End No. No. Annotation Transcript Position Position129 Chr8 TSPYL5 98289858 98290220 130 chr12 ST8SIA1 22487528 22487620131 Chr19 ZNF568 37407197 37407365 132 chr6 ELOVL2 11044395 11044834 133Chr1 cd1d 158150797 158151205 134 Chr7 ELMO1 37487755 37488477 193 Chr4BMP3 81031173 81031262 27 chr16 NDRG4 NM_020465; 58497251 58497332NM_001130487; NM_022910 101 Chr9 CDKN2A NM_000077; 21974710 21974763NM_001195132; NM_058195; NM_058197 90 Chr20 chr20.2188420.21884802188420 2188480 77 chr14 DIO3 NM_001362 1020261204 102026145 92 Chr21HUNK NM_014586 33246580 3326650

TABLE 4 Primers for DMRs Provided in Table 3. DMR No. MarkerForward Primer (5′-3′) Reverse Primer (5′-3′) 129 TSPYL5TGG CGG CGG AGG TAG TCG ATC CCG ACC GAA TTT TAA AGA TAC (SEQ IDAAC TAA CGT C (SEQ ID NO: NO: 1) 2) 130 ST8SIA1 GAC GTT TGT CGT CGGAAA AAC CCT CCG CTA GTT CGT TC (SEQ ID NO: CCA CTT CGC (SEQ ID NO: 3) 4)131 ZNF568 TTG AGA TGT TGG GTG CGC TAA CGC GAA AAA AAG GCG ATT C (SEQ IDATA ATT CGA CG (SEQ ID NO: 5) NO: 6) 132 ELOVL2 CGGTTTTATTTATTATGATCGACTACCCTAAACAACGC TCGTAGCGG (SEQ ID NO: ATCGC (SEQ ID NO: 8) 7) 133cd1d GCG CGT AGC GGC GTT CCC ATA TCG CCC GAC TC (SEQ ID NO: 9)GTA A (SEQ ID NO: 10) 134 ELMO1 TTA TAT TTT TCG TTT TTAGAA AAC CCG CCG AAA GTA ATT TCG CGT TAG C CAT TTC GA (SEQ ID NO: 12)(SEQ ID NO: 11) 193 BMP3 GTTTAATTTTCGGTTTCGT CGCTACGAAACACTCCGACGTC (SEQ ID NO: 13) (SEQ ID NO: 14)  27 NDRG4 CGGITTTCGTTCGTTITTTCCGCCTTCTACGCGACTA CG (SEQ ID NO: 15) (SEQ ID NO: 16) 101 CDKN2AGGGGCGTTGTTTAACGTA GCTACAAACCCTCTACCCA TCGAATAGTTAC (SEQ IDCCTAAATCGAC (SEQ ID NO: NO: 17) 18)  90 chr20.2188420.2TTTTAGTAAGGGTCGTATT CAAAAACTCGCTAACACGA 188480 GGACGT (SEQ ID NO: 19)AACTCCCG (SEQ ID NO: 20)  77 DIO3 GtTCGtCGttCGGGtC (SEQTCCTTCGCTaCCGAAAaCG ID NO: 21) (SEQ ID NO: 22)  92 HUNKGttTCGttACGGATtCGtC TaCTCGTaaAAaaaCGCCG (SEQ ID NO: 23) (SEQ ID NO: 24)

TABLE 5 Information for DMRs Distinguishing Between LGD, HGD and EAC,and Distinguishing Between BE and BED DMR Chromosome Gene DMR Start DMREnd No. No. Annotation Transcript Position Position 135 Chr16 NDRG458497395 58497458 90 Chr20 chr20.2188420.2188480 2188420 2188480 77chr14 DIO3 NM_001362 1020261204 102026145

TABLE 6 Information for DMRs Distinguishing BE from BED DMR ChromosomeGene DMR Start/End No. No. Annotation Transcript Positions 136 14 VSX2NM_182894 74724254-74724300 137 9 ROR2 NM_004560 94712523-94712575 138 9ROR2 NM_004560 94712480-94712521 139 1 ERO1LB NM_019891236444768-236444845 140 16 RAB11FIP3 NM_014700 476335-476351 141 15HOMER2 NM_199332; 83621577-83621602 NM_004839; NM_199330; NM_199331 14221 DSCR6 NM_018962 38379205-38379295 143 15 HOMER2 NM_199332;83621302-83621420 NM_004839; NM_199330; NM_199331 144 4 C4orf48NM_001168243; 2043778-2043860 NM_001141936 145 6 OGFRL1 NM_02457671998477-71998657 146 8 TOX NM_014729 60031838-60032005 147 2 SERPINE2NM_001136530; 224904018-224904069 NM_006216; NM_001136528 148 11 DENND5ANM_015213 9286532-9286607 149 13 TNFRSF19 NM_018647; 24153164-24153364NM_148957 150 13 TNFRSF19 NM_018647; 24152949-24153119 NM_148957 151 14CIDEB NM_014430 24780120-24780207 152 9 FBXO10 NM_01216637576336-37576403 153 13 ATP12A NM_001676; 25254666-25254800NM_001185085 154 9 CDKN2A NM_000077; 21975053-21975199 NM_001195132;NM_058195; NM_058197 155 10 STK32C NM_173575 134120900-134120935 156 19LRP3 NM_002333 33685030-33685057 157 9 NCRNA00092 NR_02412998783837-98783927 158 15 ARNT2 NM_014862 80697235-80697338 159 15 ARNT2NM_014862 80696974-80697085 160 1 HTR6 NM_000871 19991341-19991374 161 6SYNE1 NM_015293; 152623220-152623293 NM_182961; NM_033071 162 1 HTR6NM_000871 19991278-19991318 163 19 LRP3 NM_002333 33685156-33685205 1642 IGFBP2 NM_000597 217497874-217497957 165 1 MAX.chr1.244013647- —244013647-244014036 244014036 166 9 LPAR1 NM_057159; 113801112-113801189NM_001401 167 6 SYNE1 NM_015293; 152623302-152623313 NM_182961;NM_033071 168 5 MCC NM_001085377; 112630385-112630541 NM_002387 169 2SLC16A14 NM_152527 230933219-230933384 170 2 MAX.chr2.11623000- —11623000-11623066 11623066 171 3 ST3GAL6 NM_006100 98451352-98451466 17210 STK32C NM_173575 134120798-134120896 173 10 NEURL NM_004210105254137-105254241 174 2 INHBB NM_002193 121103407-121103512 175 14PRIMA1 NM_178013 94255128-94255181 176 3 ST3GAL6 NM_00610098451485-98451504 177 16 MPV17L NM_173803; 15489844-15489897NM_001128423 178 4 MAX.chr4.184718755- — 184718755-184718789 184718789179 1 TTLL7 NM_024686 84464797-84464851 180 14 PRIMA1 NM_17801394255078-94255084 181 20 DIDO1 NM_033081; 61560714-61560835NM_001193369; NM_022105; NM_080797; NM_001193370; NM_080796 182 4C4orf31 NM_024574 121992630-121992757 183 16 IRX3 NM_02433654320149-54320196 184 19 LRP3 NM_002333 33685073-33685127 185 11 PRR5LNM_001160167; 36398162-36398218 NM_001160168; NM_024841 186 3 ST3GAL6NM_006100 98451114-98451159 187 5 MAX.chr5.60921709- — 60921709-6092180860921808

TABLE 7 Information for DMRs Distinguishing BE from normal tissue DMRChromosome Gene DMR Forward MSP Reverse MSP No. No. AnnotationCoordinates Primer (5′-3′) Primer (5′-3′) 188  7 adcy1 45613877-GGT TCG GTT CCG ACC GTA ATC 45614572 GTC GTA GCG C CTC GAC GA (SEQ(SEQ ID NO: 25) ID NO: 26) 189  7 LRRC4 127671993- GTT AAT TTCCGT AAT ACA ATA 127672310 GCG AGG TAG CTC TTA TAT ATT GCG ACG (SEQAAC GCC GCT ID NO: 27) (SEQ ID NO: 28) 190 19 ZNF569 37957760-TGT GGA ATC CCC ACC CAA 37958046 GGG GTT TGT CAC AAA AAA TCCGTT CGC (SEQ ID GAC G (SEQ ID NO: 29) NO: 30)  21 19 ZNF682 20149796-GGA GTT TAT CCC CGC AAT 20149923 TTT GGG AAG CGA AAC AAA CG AGT CGC (SEQ(SEQ ID NO: 32) ID NO: 31) 191 14 PTGDR 52735290- GGG TAG AGAACT AAA TCA CCT 52735389 ATA TAT AGT CCT ACT ACT AAC GAA GAG TACGCT (SEQ ID NO: GG (SEQ ID NO: 34) 33) 192 10 SFMBT2 7452029-GCG ACG TAG CCA ACG CGA 7452452 TCG TCG TTG T AAA AAA CGC G(SEQ ID NO: 35) (SEQ ID NO: 36)

Example II

This example describes the materials and methods utilized in generatingTables 1 and 7.

18 Barrett's esophagus (BE) and 18 normal esophagus tissue samples wereselected from institutional cancer registries at Mayo Clinic Rochesterand were reviewed by an expert pathologist to confirm correctclassification. Normal leukocyte controls were provided by the MayoBiospecimens Linking Investigators and Clinicians to GIH Cell SignalingResearch Clinical Core.

Library Preparation: Genomic DNA (300 ng) was fragmented by digestionwith 10 Units of MspI, a methylation-specific restriction enzyme whichrecognizes CpG-containing motifs, to enrich sample CpG content andeliminates redundant areas of the genome.

Digested fragments were end-repaired and A-tailed with 5 Units of Klenowfragment (3′-5′ exo-), and ligated overnight to methylated TruSeqadapters (Illumina, San Diego Calif.) containing barcode sequences (tolink each fragment to its sample ID.) Size selection of 160-340 bpfragments (40-220 bp inserts) was performed using Agencourt AMPure XPSPRI beads/buffer (Beckman Coulter, Brea Calif.). Buffer cutoffs were0.7×-1.1× sample volumes of beads/buffer. Final elution volume was 22 uL(EB buffer—Qiagen, Germantown Md.); qPCR was used to gauge ligationefficiency and fragment quality on a small sample aliquot. Samples thenunderwent bisulfate conversion (twice) using a modified EpiTect protocol(Qiagen). qPCR and conventional PCR (PfuTurbo Cx hotstart—Agilent, SantaClara Calif.) followed by Bioanalyzer 2100 (Agilent) assessment onconverted sample aliquots determined the optimal PCR cycle number priorto final library amplification. The following conditions were used forfinal PCR: 1.) each 50 uL reaction contained 5 uL of 10× buffer, 1.25 uLof 10 mM each deoxyribonucleotide triphosphate (dNTP), 5 uL primercocktail (˜5 uM), 15 uL template (sample), 1 uL PfuTurbo Cx hotstart and22.75 water; temperatures and times were 95 C-5 min; 98 C-30 sec; 16cycles of 98 C-10 sec, 65 C-30 sec, 72 C-30 sec, 72 C-5 min and 4 Chold, respectively. Samples were combined (equimolar) into 4-plexlibraries based on the randomization scheme and tested with thebioanalyzer for final size verification, and with qPCR using phiXstandards and adaptor-specific primers.

Sequencing and Bioinformatics: Samples were loaded onto flow cellsaccording to a randomized lane assignment with additional lanes reservedfor internal assay controls. Sequencing was performed by the NextGeneration Sequencing Core at the Mayo Clinic Medical Genome Facility onthe Illumina HiSeq 2000. Reads were unidirectional for 101 cycles. Eachflow cell lane generated 100-120 million reads, sufficient for a mediancoverage of 30-50 fold sequencing depth (read number per CpG) foraligned sequences. Standard Illumina pipeline software called bases andsequenced read generation in the fastq format. As described previously,(28) SAAP-RRBS, a streamlined analysis and annotation pipeline forreduced representation bisulfate sequencing, was used for sequencealignment and methylation extraction.

MSP Primer design: Primers for 6 top markers from the sequencing resultswere designed and ordered (IDT, Coralville Iowa) to target specificbisulfite-modified methylated sequences (table 7). The designs were doneby either Methprimer software (University of California, San FranciscoCalif.) or MSPPrimer (Johns Hopkins University, Baltimore, Md.). Assayswere tested and optimized by qPCR with SYBR Green on dilutions ofuniversally methylated and unmethylated genomic DNA controls.

Methylation specific PCR: Quantitative MSP reactions were performed onindependent tissue-extracted DNA: 108 BE samples—36 with high gradedysplasia, 36 with low grade dysplasia, and 36 with no dysplasia, 18normal esophagus samples, and 36 normal leukocyte samples.

Statistical Analysis: Candidate CpGs were filtered by a prioriread-depth and variance criteria, significance of differential%-methylation percentages between cases and controls and discriminationof cases from controls based on area under the receiver operatingcharacteristics curve (AUC) and target to background ratio.

For the RRBS discovery phase, the primary comparison of interest was themethylation difference between Barrett's cases, esophagus controls andleukocyte controls at each mapped CpG. CpG islands are biochemicallydefined by an observed to expected CpG ratio >0.6·(30) However, for thismodel, tiled units of CpG analysis “differentially methylated region(DMR)” were created based on distance between CpG site locations foreach chromosome. Islands with only single CpGs were excluded. IndividualCpG sites were considered for differential analysis only if the totaldepth of coverage per disease group was ≥200 reads (an average of 10reads/subject) and the variance of %-methylation was >0 (non-informativeCpGs were excluded). Read-depth criteria were based on the desiredstatistical power to detect a 10% difference in the %-methylationbetween any two groups in which the sample size of each group was 18individuals. Statistical significance was determined by logisticregression of the methylation percentage per DMR, based on read counts.To account for varying read depths across individual subjects, anover-dispersed logistic regression model was used, where dispersionparameter was estimated using the Pearson Chi-square statistic of theresiduals from fitted model. DMRs, ranked according to theirsignificance level, were further considered if %-methylation in benignesophagus and leukocyte controls, combined, was ≤1% but ≥10% inBarrett's cases. This resulted in 78 markers (Table 1). All had AUCsgreater than 0.90 and fold changes greater than 25.

For the 6 marker qMSP validation study (Table 7), the primary outcomewas the area under the receiver operating characteristics curve (AUC)for each marker, as calculated from logistic regression models of the %methylated copy number per sample with BE in comparison to normalesophagus and normal leukocytes. For each marker, AUCs were again >0.90and the quantitative difference in mean values of candidate genomic copynumber per sample between cases and controls were at least 50-fold.

Example III

This example describes the materials and methods utilized in generatingTables 2 and 6.

36 Barrett's esophagus with high and low dysplasia (BED) and 18Barrett's esophagus with no dysplasia (BE) tissue samples were selectedfrom institutional cancer registries at Mayo Clinic Rochester and werereviewed by an expert pathologist to confirm correct classification. 18Normal leukocyte controls were provided by the Mayo Biospecimens LinkingInvestigators and Clinicians to GIH Cell Signaling Research ClinicalCore.

Library Preparation: Genomic DNA (300 ng) was fragmented by digestionwith 10 Units of MspI, a methylation-specific restriction enzyme whichrecognizes CpG-containing motifs, to enrich sample CpG content andeliminates redundant areas of the genome. Digested fragments wereend-repaired and A-tailed with 5 Units of Klenow fragment (3′-5′ exo-),and ligated overnight to methylated TruSeq adapters (Illumina, San DiegoCalif.) containing barcode sequences (to link each fragment to itssample ID.) Size selection of 160-340 bp fragments (40-220 bp inserts)was performed using Agencourt AMPure XP SPRI beads/buffer (BeckmanCoulter, Brea Calif.). Buffer cutoffs were 0.7×-1.1× sample volumes ofbeads/buffer. Final elution volume was 22 uL (EB buffer—Qiagen,Germantown Md.); qPCR was used to gauge ligation efficiency and fragmentquality on a small sample aliquot. Samples then underwent bisulfateconversion (twice) using a modified EpiTect protocol (Qiagen). qPCR andconventional PCR (PfuTurbo Cx hotstart—Agilent, Santa Clara Calif.)followed by Bioanalyzer 2100 (Agilent) assessment on converted samplealiquots determined the optimal PCR cycle number prior to final libraryamplification. The following conditions were used for final PCR: 1.)each 50 uL reaction contained 5 uL of 10× buffer, 1.25 uL of 10 mM eachdeoxyribonucleotide triphosphate (dNTP), 5 uL primer cocktail (˜5 uM),15 uL template (sample), 1 uL PfuTurbo Cx hotstart and 22.75 water;temperatures and times were 95 C-5 min; 98 C-30 sec; 16 cycles of 98C-10 sec, 65 C-30 sec, 72 C-30 sec, 72 C-5 min and 4 C hold,respectively. Samples were combined (equimolar) into 4-plex librariesbased on the randomization scheme and tested with the bioanalyzer forfinal size verification, and with qPCR using phiX standards andadaptor-specific primers.

Sequencing and Bioinformatics: Samples were loaded onto flow cellsaccording to a randomized lane assignment with additional lanes reservedfor internal assay controls. Sequencing was performed by the NextGeneration Sequencing Core at the Mayo Clinic Medical Genome Facility onthe Illumina HiSeq 2000. Reads were unidirectional for 101 cycles. Eachflow cell lane generated 100-120 million reads, sufficient for a mediancoverage of 30-50 fold sequencing depth (read number per CpG) foraligned sequences. Standard Illumina pipeline software called bases andsequenced read generation in the fastq format. As described previously,(28) SAAP-RRBS, a streamlined analysis and annotation pipeline forreduced representation bisulfate sequencing, was used for sequencealignment and methylation extraction.

MSP Primer design: Primers for the top 66 markers from the sequencingresults were designed and ordered (IDT, Coralville Iowa) to targetspecific bisulfite-modified methylated sequences (table 7). The designswere done by either Methprimer software (University of California, SanFrancisco Calif.) or MSPPrimer (Johns Hopkins University, Baltimore,Md.). Assays were tested and optimized by qPCR with SYBR Green ondilutions of universally methylated and unmethylated genomic DNAcontrols.

Methylation specific PCR: Quantitative MSP reactions were performed onindependent tissue-extracted DNA: 108 BE samples—36 with high gradedysplasia, 36 with low grade dysplasia, and 36 with no dysplasia, 18normal esophagus samples, and 36 normal leukocyte samples.

Statistical Analysis: Candidate CpGs were filtered by a prioriread-depth and variance criteria, significance of differential%-methylation percentages between cases and controls and discriminationof cases from controls based on area under the receiver operatingcharacteristics curve (AUC) and target to background ratio.

For the RRBS discovery phase, the primary comparison of interest was themethylation difference between Barrett's cases with high and low gradedysplasia, Barrett's with no dysplasia controls, and leukocyte controlsat each mapped CpG. CpG islands are biochemically defined by an observedto expected CpG ratio >0.6·(30) However, for this model, tiled units ofCpG analysis “differentially methylated region (DMR)” were created basedon distance between CpG site locations for each chromosome. Islands withonly single CpGs were excluded. Individual CpG sites were considered fordifferential analysis only if the total depth of coverage per diseasegroup was ≥200 reads (an average of 10 reads/subject) and the varianceof %-methylation was >0 (non-informative CpGs were excluded). Read-depthcriteria were based on the desired statistical power to detect a 10%difference in the %-methylation between any two groups in which thesample size of each group was 18 individuals. Statistical significancewas determined by logistic regression of the methylation percentage perDMR, based on read counts. To account for varying read depths acrossindividual subjects, an over-dispersed logistic regression model wasused, where dispersion parameter was estimated using the PearsonChi-square statistic of the residuals from fitted model. DMRs, rankedaccording to their significance level, were further considered if%-methylation in benign esophagus and leukocyte controls, combined, was≤1% but ≥10% in Barrett's cases. This resulted in 57 markers (Table 2).All had AUCs between 0.60 and 0.87 and fold changes between 2 and 10. Asecond sorting of the data was performed, loosening the islandrestriction metrics and focusing on groupings of highly discriminatesingle CpGs. A batch to batch effect on the leukocyte controls was alsoremoved which increased the overall coverage. This resulted in 52additional markers with increased fold changes (7-52) and similar AUCs(Table 6).

For the 66 DMR qMSP validation study, the primary outcome was the areaunder the receiver operating characteristics curve (AUC) for eachmarker, as calculated from logistic regression models of the %methylated copy number per sample with BED in comparison to BE andnormal leukocytes. 12 markers demonstrated superior performance (Table3). AUCs were 0.86-0.97 and fold changes 2-24. 10 of the 12 markers,along with BMP3 and NDRG4, were carried into the esophageal brushingfeasibility study.

Example IV

This example describes the materials and methods utilized in generatingTables 3, 4 and 5.

Consenting BE subjects scheduled for endoscopic BE surveillance orendoscopic assessment of BE related cancers underwent whole esophagealbrushings using a high capacity cytology brush (Hobbs Medical, StaffordSprings, Conn.) with circumferential sampling from the cardia throughthe full esophageal length (BE+squamous mucosa). The cytology brush wasremoved from the handle and placed into a vial of stability/cell lysissolution and frozen until processing. DNA was extracted using the GentraPuregene Buccal procedure (Qiagen, Valencia, Calif.). 2 ug of DNA fromeach patient sample was treated with sodium bisulfite and purified usingthe EZ DNA Methylation kit (Zymo Research, Irvine, Calif.). MSP wasperformed using 20 ng of converted DNA on the 10 of the 12 validatedDMRs from the BED vs. BE study (Table 3). The primer sequences arehighlighted in Table 4. In addition the BMP3 and NDRG4 Cologuard QuARTsassays were run. The method of DeLong, DeLong and Clarke-Pearson wasused to compare AUCs and measure significance of differences. ABonferroni correction was used to avoid bias from multiple comparisons.The 3 markers which (in combination) demonstrated the highestdiscrimination for BED vs. BE are listed in Table 5.

Example V

This example demonstrates the discovery, validation and feasibilitytesting of methylated DNA markers for detection of Barrett's Esophagus.

Phase 1 Methods and Results:

Pathologist verified FFPE tissues were provided by the Mayo ClinicTissue Registry. Clinical groups consisted of patients with Barrett'sHGD (N=34), Barrett's LGD (N=34), Barrett's no dysplasia (N=34),esophageal adenocarcinomas (N=12), esophageal squamous cell carcinomas(N=12), normal cardia (N=13), normal esophagus (N=25). DNA was purifiedusing the Qiagen Mini kit and quantified by absorbance and picogreenanalysis. Bisulfite conversion was performed using the Zymo method.Methylation markers consisted of top candidates from 3 categories ofRRBS subsets: 1) 45 BED vs. BE DMRs (differentially methylated regions),2) 5 BE vs. normal esophagus DMRs, and 3) 33 previously validatedesophageal cancer markers. (Note: All of these DMRs were previouslyfiltered against normal leukocytes for <1% background methylation.)Methylation specific PCR (MSP) primers were designed for each of thesegenomic regions and tested on 3 sets of methylation controls forperformance. Table 8 provides DMR information including chromosomenumber, gene annotation, and DMR start/stop position for such markersidentified to separate BE from normal tissue. QMSP (SYBR Green) wasperformed using Roche 480 LightCyclers. Serially diluted universalmethylated DNA was used as a standard. In addition, QuARTs assays wererun on the markers BMP3, NDRG4, SFMBT2, and VAV3. These latter 4 include2 reference genes β-actin and ZDHHC1 in their triplex assay formats.

Results were normalized against β-actin and ZDHHC1 and analyzedlogistically in JMP. Areas under the ROC curve (AUC) were calculatedalong with fold changes and p-values. Performance cut-offs for phase 2were AUC≥0.95, fold change ≥25, and p-value ≤0.1. 13 markers passedthese criteria: CDKN2A, SFMBT2, VAV3, D103, ELMO1, FEM1B, HUNK, ADCY1,CD1D, ST3GAL6, LRRC4, NDRG4, and BMP3 (Table 9 provides the identity andprimer sequences for these assays including OPLAH).

TABLE 8 Information for DMRs distinguishing BE and normal tissue DMRGene Chromosome DMR Start and End No. Annotation No. Position 2 ZNF56819 37407197-37407284 3 IRX4 5 1883238-1883312 4 RGS17 6153451813-153451881 6 EPHA5 4 66536122-66536220 7 SFMBT2.1869 107451771-7451869 14 A1BG 19 58859193-58859258 30 DPY19L2 1264061896-64062007 77 DIO3 14 102026104-102026145 80 WNT2 7116964596-116964659 82 RGL3 19 11529371-11529430 83 FEM1B 1568569729-68569799 84 ARNT2 15 80696170-80696177 85 LARP6 1571146759-71146820 86 ZC3HAV1L 7 138720915-138720957 88Max.chr15.41877531.41877548 15 41877531-41877548 90Max.chr20.2188420.2188480 20 2188420-2188480 91 KLHL14 1830351268-30351486 92 HUNK 21 33246580-33246650 93 LOC100131691 1959073783-59073952 94 Max.chr13.95620964.95621061 13 95620964-95621061 95Max.chr5.926920.927009 5 926920-927009 96 C6orf114 6 13488436-1348853097 ARHGEF10 8 1771362-1771477 98 VSTM2L 20 36531194-36531312 99 ACAD11 3132378234-132378296 100 WSB2 12 118500206-118500305 101 CDKN2A 921974710-21974763 102 Max.chr6.27064706.27064783 6 27064706-27064783 108WIF1 12 65514995-65515089 122 DLX5 7 96653893-96653955 135 NDRG4 1658497395-58497451 136 VSX2 14 74724254-74724300 141 HOMER2 1583621577-83621602 142 DSCR6 21 38379205-38379295 144 C4orf48 42043778-2043860 146 TOX 8 60031838-60032005 148 DENND5A 119286532-9286607 149 TNFRSF19 13 24153164-24153364 152 FBXO10 937576336-37576403 154 CDKN2A 9 21975053-21975199 156 LRP3 1933685030-33685057 164 IGFBP2 2 217497874-217497957 166 LPAR1 9113801112-113801189 171 ST3GAL6 3 98451352-98451466 173 NEURL 10105254137-105254241 175 PRIMA1 14 94255128-94255181 178MAX.chr4.184718755-184718789 4 184718755-184718789 179 TTLL7 184464797-84464851 181 DIDO1 20 61560714-61560835 185 PRR5L 1136398162-36398218 187 MAX.chr5.60921709-60921808 5 60921709-60921808 193BMP3 4 81952348-81952402 194 VAV3 1 108507608-108507679 195 CYP26C1.F 1094822416-94822607 196 EMX1.F 2 73147710-73147772 197 LOC645323.R 725896389-25896501 198 ELOVL2.F 6 11044395-11044834 199 FLI1.F 11128563956-128564209 200 KCNK12 2 47797187-47797452 201 SFMBT2.893 107450242-7450831 202 SFMBT2.895 10 7452029-7452452 203 ZNF625.F 1912267378-12267677 204 ELMO1.F 7 37487755-37488477 205 ST8SIA1.F 1222487528-22487620 206 ZNF568.R 19 37407197-37407365 207 GRIN2D.R 1948918144-48918350 208 TBX15.F 1 119527066-119527655 209 TSPYL5.F 898289858-98290220 210 ZNF610.R 19 52839503-52840013 211 ZNF671.F 1958238810-58238955 212 ZNF781.F 19 38182950-38183127 213 ADCY11 745613877-45614572 214 C13orf18 13 46960767-46961669 215 CD1D 1158150797-158151205 216 AK055957 (chr12.133) 12 133484978-133485739 217CLEC11A 19 51228217-51228732 218 RSPO3 6 127440492-127441039 219 TOX2 2042544780-42544835 220 VWC2 7 49813135-49814168 221 DOCK10.F 2225907226-225907322 222 LRRC4.R 7 127671993-127672310 223MAX.chr11.123301058.123301255.R 11 123301058-123301255 224 STC.ZNF569.R19 37957760-37958046 225 ZNF682.R 19 20149796-20149923 226 GRM8.F 7126891703-126892479 227 PTGDR.R 14 52735290-52735389 228 OPLAH 8145106349-145106456 229 SFMBT2 10 7452885-7452956

TABLE 9 Primers for specific DMRs Provided Described in Example V. DMRForward Primer Reverse Primer No. Marker (5′-3′) (5′-3′) Probe 204ELMO1.F TTA TAT TTT TCG GAA AAC CCG CCG TTT TTA GTA ATT AAA CAT TTC GATCG CGT TAG C (SEQ ID NO: 12) (SEQ ID NO: 11) 213 ADCY1 GGT TCG GTT GTCCCG ACC GTA ATC GTA GCG C (SEQ ID CTC GAC GA (SEQ NO: 25) ID NO: 26) 215CD1D GCG CGT AGC GGC CCC ATA TCG CCC GTT TC (SEQ ID NO:GAC GTA A (SEQ ID 9) NO: 10) 222 LRRC4.R GTT AAT TTC GCG CGT AAT ACA ATAAGG TAG GCG ACG CTC TTA TAT ATT (SEQ ID NO: 27) AAC GCC GCT (SEQID NO: 28) 228 OPLAH TGC GTA GGT GAT ACA AAA CAC ATC AGG GAG GGG TTACTA TTA ACG CGA A C (SEQ ID NO: 47) (SEQ ID NO: 48) 101 CDKN2AGGGGCGTTGTTTAA GCTACAAACCCTCT CGTATCGAATAGTTA ACCCACCTAAATCGC (SEQ ID NO: 17) AC (SEQ ID NO: 18)  77 DIO3 GtTCGtCGttCGGGtCTCCTTCGCTaCCGA (SEQ ID NO: 21) AAaCG (SEQ ID NO: 22)  83 FEM1BtTtttAtATTTCGGGAAt TaAaCCGaaaTTAaaa TtAGAAACGtC (SEQ AAaaaaTTaCGCGID NO: 49) (SEQ ID NO: 50)  92 HUNK GttTCGttACGGATtCG TaCTCGTaaAAaaaCtC (SEQ ID NO: 23) GCCG (SEQ ID NO: 24) 171 ST3GAL6 GTTTCGTTCGAAAGCGAATCTCCCGAAA GTAGGGGTTCG AATAAAACGTT (SEQ (SEQ ID NO: 37) ID NO: 38)193 BMP3 GTTTAATTTTCGGTT CGCTACGAAACACT CGCCGAGGCGG TCGTCGTC (SEQ IDCCGA (SEQ ID NO: TTTTTTGCG NO: 13) 14) (SEQ ID NO: 43) 135 NDRG4CGGTTTTCGTTCGTT CCGCCTTCTACGCG CCACGGACGGT TTTTCG (SEQ ID NO:ACTA (SEQ ID NO: TCGTTTATCG 15) 16) (SEQ ID NO: 44) 194 VAV3TCGGAGTCGAGTTT CGAAATCGAAAAAA CGCCGAGGCGG AGCGC (SEQ ID NO:CAAAAACCGC (SEQ CGTTCGCGA 39) ID NO: 40) (SEQ ID NO: 45) 229 SFMBT2GTCGTCGTTCGAGA CGAACAAAAACGAA CCACGGACGAT GGGTA (SEQ ID NO:CGAACGAA (SEQ ID CGGTTTCGTT 41) NO: 42) (SEQ ID NO: 46)

Phase 2 Methods and Results:

49 cases with and 36 controls without BE were recruited prior toendoscopy. Median age was 69 (range 63-73) and 59 (45-67) and mencomprised 92% and 42%, respectively. BE cases had >1 cm (median=2 cm;IQR 4-8) of circumferential columnar mucosa with confirmed intestinalmetaplasia; controls had no BE endoscopically. Specimens were obtainedusing a high capacity endoscopic cytology brush (Hobbs Medical, StaffordSprings Conn.); the cardia, BE (in cases), and full esophageal lengthwere brushed to simulate a swallowed sponge sampling device. The brushwas placed in a 2 ml vial containing lysis buffer and promptly frozen at−80 C. Samples were thawed and processed as a batch in blinded fashion.Following vigorous vortexing of the vial to remove all cellular materialfrom the brush, DNA was purified using the Gentra Puregene kit (Qiagen).This method allows for the simultaneous harvesting of both free andcellular DNA. Samples were then treated with sodium bisulfite andrecovered using the EZ DNA methylation kit (Zymo Research).

Methylation of the 13 target genes was assayed by QMSP and QuARTs asbefore on Roche 480 LightCyclers. β-actin and ZDHHC1 were alsoquantified as markers for total human DNA. Several markers (e.g. BMP3,CDKN2A, CD1D, HUNK, ELMO1, DIO3) showed exceptional discrimination forBE with AUCs 0.91-0.97; methylation level distributions from BE casesand controls differed substantially (FIG. 2). Methylation levelscorrelated with BE length and presence of dysplasia, p<0.05. FIG. 3shows a hit matrix of top methylated DNA markers from Phase 2highlighting complementarity (endoscopic brush study). 10 Barrett'sspecific markers were chosen for phase 3 testing: BMP3, NDRG4, VAV3,SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH. OPLAH was notincluded in the earlier phases, but was added here due to its excellentperformance in discriminating esophageal cancers from normal tissue.

Phase 3 Methods and Results:

A capsule sponge device (EsophaCap, Capnostics) was swallowed andwithdrawn in 10 cases with BE and 12 controls without apparent BEfollowed by endoscopy within 24 hours. Among 10 cases and 12 controls,median age was 65 (59-69) and 40 (34-61) and men comprised 70% and 45%,respectively. Median BE length was 4.5 cm (IQR 2-9). The device was thenplaced in a vial containing 20 mL of cell preservation buffer(PreservCyt). Samples were vortexed and transferred into a 50 mLcentrifuge tube. This step was repeated with an additional aliquot ofPreservCyt for a total of 40 ml. The cells were pelleted and lysed in 1mL of buffer (Puregene Buccal Cell Kit) and extracted following themanufacturer's directions. A second extraction method (Maxwell—Promega)was also tested. Following bisulfite conversion (Zymo Research), thesamples were assayed by QPCR as before. Distributions of top markersfrom the sponge were highly discriminant for BE. At 100% specificity, apanel of markers detected all 9 BE cases (1 did not meet inclusioncriteria) meeting inclusion criteria (100% sensitivity).

FIG. 4 shows methylated DNA marker levels (PCR copies/30 ng DNA) in BEcases and normal (Nl) controls from Phase 3 (capsule sponge study).

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inpharmacology, biochemistry, medical science, or related fields areintended to be within the scope of the following claims.

1. (canceled)
 2. A method comprising: extracting genomic DNA from abiological sample of a human individual suspected of having or havinggastro-intestinal neoplasm, treating the extracted genomic DNA withbisulfite, amplifying the bisulfite-treated genomic DNA using primersspecific for one or more markers selected from BMP3, NDRG4, VAV3,SFMBT2, DIO3, HUNK, ELMO1, CD1D, CDKN2A, and OPLAH, wherein the one ormore markers at least includes ELMO1, measuring the methylation level ofthe CpG site for the markers by methylation-specific PCR, quantitativemethylation-specific PCR, methylation-sensitive DNA restriction enzymeanalysis, or bisulfite genomic sequencing PCR.
 3. The method of claim 2,wherein the biological sample comprises cells, tissues, and/orsecretions from the upper gastrointestinal tract and/or the lowergastrointestinal tract.
 4. The method of claim 2, wherein the biologicalsample is a stool sample, a tissue sample, a blood sample, or a urinesample.
 5. A method comprising: extracting genomic DNA from a biologicalsample of a human individual suspected of having or havinggastro-intestinal neoplasm, treating the extracted genomic DNA withbisulfite, amplifying the bisulfite-treated genomic DNA using primersspecific for no more than ten markers, wherein the no more than tenmarkers include either ZNF682 or ZNF682 and NDRG4, measuring themethylation level of the CpG site for the markers bymethylation-specific PCR, quantitative methylation-specific PCR,methylation-sensitive DNA restriction enzyme analysis, or bisulfitegenomic sequencing PCR.
 6. The method of claim 5, wherein one of the nomore than ten markers is VAV3.
 7. The method of claim 5, wherein thebiological sample comprises cells, tissues, and/or secretions from theupper gastrointestinal tract and/or the lower gastrointestinal tract. 8.The method of claim 5, wherein the biological sample is a stool sample,a tissue sample, a blood sample, or a urine sample.